Fluorescence microscopy

BT-2062 (Lecture 4)
14th August 2023
Santhosh Sethuramanujam
Last Class…
• Bright field microscopy
• Scattering
• Biological samples not good
• Phase contrast microscopy
• Phase shifting of light in accordance to the
sample thickness
• Optical interference
• DIC microscopy
• Similar to Phase contrast
• Requires polarization optics.
• Apparent 3D image
• Phase plate properties
• More linear signal • Why not use a dye to visualize cells in bright field?
• Why is the 3D image apparent?
DIC principle
• How do you manipulate the
phase shifts? Like seen in
phase contrast
microscopy….
Nomarski prism
• The two beams have different path
lengths in the prism.
• Ordinary ray – passes through
normally
• Extra-ordinary ray – different
polarization greater distance. Hence
the two beams are phase shifted.
• This can be enhanced by the angle of
incidence.
Today
• Conventional fluorescence microscopy
• Confocal microscopy
• 2-photon excitation microscopy.
Fluorescence
• Property of molecules to
absorb photons of a specific
wavelength, say UV.
• Emits a photon at longer
wavelength, say visible light.
• Such molecules are tagged
to bio-molecules to track
their location in a cell,
tissue, etc.
Stokes shift
• Range of wavelengths over
which the molecule absorbs and
emits photons.
• Emission (fluorescence)
wavelength higher than the
absorbance.
Instrumentation –
bright field microscope
• Light source
• Condenser
• Increases numerical aperture
• Sample (stage)
• Objective
• magnification
• Eyepiece
• Magnification.
Instrumentation –
fluorescence microscope
• Light source
• Excitation same path as emission.
• Condenser
• Sample (stage)
• Objective
• Acts a condenser and a objective
• magnification
• Eyepiece
• Magnification.
Dichroic mirror

• Mirror reflects only specific

wavelengths, in this case, the
excitation wavelength.
• The emission wavelength is
not reflected, and reaches
the detector.
Fluorescence microscopy
• Spatial localization of proteins
• PKCalpha expression in bipolar
cells of zebrafish retina.
• The protein is labelled using
antibodies against PKCalpha
(more later in the course on
labeling)
• Notice the diffuse green
illumination, which reduces the
contrast of the image
• in particular, a problem in thick
samples.
Laser Scanning confocal microscopy

If interested in seeing this in action, let me know…

Disadvantage of widefield fluorescence…
• The excitation illuminates
the whole sample,
consequently, even
fluorophores from above
and below emit photons.
• Out-of-focus plane photons
cause blurring of the image.

• Confocal imaging eliminates

the out-of-focus signals.
• Optically section the tissue
Confocal microscopy principle.
• Use pinholes to
eliminate the out of
focus light.

• Laser source is used

 focused high
intensity beam
• Passed through a
pinhole.
• Excitation in x-y plane
is limited
Confocal microscopy principle.
• The emitted photons will
be from the small spot in
x-y plane
• Photons emitted in z will
not be localized, as above
and below will be excited.
• Second pinhole is placed
at the focal point of the
objective.
• only light coming from
the focal point on the
specimen is passed
through.

Can resolution be increased by making the pinhole smaller? Why do you need a laser for stimulation?
 How do you image beyond the focal spot?

• Galvanometer  electricity to mechanical movement.

• Mirrors to move the spot in x and y axis.
• The specimen is scanned pixel by pixel, line by line.
Laser scanning confocal microscope
• Laser pinhole galvo mirrors
 excites specimen  emission
pinhole detector.

• Confocal  because the pinhole is

place at the focus of the objective

• Laser is scanned spot by spot in a

confocal microscope.
Detector – Phototubes
• Cathode shows photoelectric effect. i.e. when
photons strike the cathode, it will eject
electron.
• Ejected electron travels to the anode in a
vacuum chamber (~90 V).
• When the electrode reaches the anode,
current flows.
• Generally, current is low. Needs to be
amplified

Why do you need vacuum?

Confocal laser scanning microscopy
• Laser through pinhole
• Dichroic that reflects laser to the galvo
(X,Y scanning unit)
• Objective  focus beam at focal point
• Emission from sample focused by
objective at the confocal pinhole
• Dichroic mirror is transparent to the
emission.
• Photons focused at pinhole enters
PMT.
• Notice how all of this is synchronized
using computers.
2-photon excitation for microscopy
• Uses concept of 2 photon
absorption
• Near simultaneous (10-15s)
absorption of 2 photons of longer
wavelength, say 720nm equal to
the energy of 360nm
• 360nm excites the fluorophore.
2-photon excitation for
microscopy
• Since 2photon absorption requires
near simultaneous incidence, it
occurs only at the point of focus.
• Fluorescence rapidly drops from the
focal distance.
• Laser passed through a fluorescent
substance. Notice the Spot in 2P vs
column in 1P.
• Hence, pinholes are not needed to
remove out of focus light, as seen in
confocal microscopy.
What is the advantage of 2-photon excitation?
• Deep penetration into tissues by
longer wavelength
• Scattering is inversely proportional to
wavelength (recall why the sky is blue)

• Non-invasive imaging strategies to

monitor deeper tissues. Best used in
the brain
Greater depth penetration
• Bottom – 2P excitation
• Top – confocal (1P excitation)
• 20 microns  signal down to half
• Signal more than 100 microns
Cortical depth
• Imaging upto 2
mm deep in the
cerebral cortex.
• Non-invasive
method giving
single
neuron/dendrite
resolution.
• Nothing comes
close.
Neuronal activity in intact tissue.
• Activity of cortical neurons
• Somas, dendrite, even spines clearly seen.
• Change in fluorescence indicate the
neuronal activity.
 Point spread function

Comparisons
widefield 2P confocal

Confocal
• X-Y Resolution is higher –why?
• Sample bleaching an issue.
• Alignment is tricky.

2-photon excitation
• Z resolution is higher – used in
thicker samples/ live tissue
imaging
• Laser is expensive
Summary
• Conventional fluorescence microscopy
• Fluorescence
• Instrumentation
• Confocal microscopy
• Principle – confocal pinhole
• Laser scanning
• 2-photon excitation microscopy.
• Principle
• Advantages - Greater penetration
• Pros and cons.
Next class…
• Super Resolution Microscopy
• Electron Microscopy