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T. C. Jagadale and S.-W. Chu
paths decided the FWHM of illumination and detection PSF that gave 4–7-fold
increase in axial resolution.
(b) Non-uniform illumination: In 2000, Gustafsson [17] demonstrated that by illuminating the sample with patterned excitation light, called Moiré pattern, lateral
resolution could be enhanced by two times, that is about 100 nm. This is called
structured illumination microscopy (SIM).
(c) Nonlinear absorption and emission: In the late 1990s, Hell [18] developed the
methods to control the behaviours of fluorophores wherein all fluorophores are
switched off except the centremost fluorophores within the diffraction-limited
illumination volume of a laser scanning microscope, resulting in development of
stimulated emission depletion (STED) microscopy with typical lateral resolution as high as 30–50 nm (can reach 6 nm with nano-diamond). During the same
period, William Moerner demonstrated [19] that how certain mutants of green
fluorescent protein showed remarkable ‘blinking’ behaviour in their individual
fluorescence emission. After several rounds of blinking, these molecules would
go into stable dark state, and could be recovered by a short burst of UV light. This
idea had fascinated Betzig [20] to develop super-resolution microscope based
on blinking fluorophores and principle of localisation. The demonstration of
controlled on/off switching of fluorescent proteins had led to the discovery of
PALM with a resolution as high as 10 nm. It is important to note that all these
far-field super-resolution imaging techniques are all based on fluorescence as a
contrast mode.
In any fluorescence microscope, the high energy photon (from lamp or laser) excites
fluorophores in the specimen and the excited fluorophore subsequently emits its own
lower energy photon. The high-contrast imaging is achieved by using spectral filtering
of fluorescence from the excitation light. Imaging protein expression, localisation,
and activity in living cells is possible with the development of genetically encoded
fluorescent proteins [21, 22]. The high specificity of fluorescence light microscopy
facilitates the collection of spatial and functional information about bio-structures
using labelled molecules. In this way, the majority of light microscopy applications
in life sciences today use fluorescence as a dominating contrast mode.
However, the widely used fluorescence microscopy has certain shortcomings that
hinder further growth of the field. Some of these are: (1) Photo-bleaching: the fluorescent molecule on excitation over a period of time accumulates electrons in its excited
state that often leads to a chemical damage of fluorophore, resulting in loss of fluorescence, that is photo-bleaching. Photo-bleaching severely limits the time over which
a sample can be observed under microscope. (2) Photo-toxicity: this effect often
occurs as fluorescent molecules under intense illumination, which have a tendency
to generate reactive chemical species. Live cells are susceptible to them, resulting in
cell mortality. (3) Low photo-stability: for super-resolution imaging based on on/off
switching, typical fluorescent labels are not strong enough to undergo the switching
cycle for multiple times, thus limiting the length of total acquisition time. (4) Autofluorescence: This reduces contrast by forming an image background that even the
best excitation/emission filters could not completely get rid of. These shortcomings
T. C. Jagadale and S.-W. Chu
paths decided the FWHM of illumination and detection PSF that gave 4–7-fold
increase in axial resolution.
(b) Non-uniform illumination: In 2000, Gustafsson [17] demonstrated that by illuminating the sample with patterned excitation light, called Moiré pattern, lateral
resolution could be enhanced by two times, that is about 100 nm. This is called
structured illumination microscopy (SIM).
(c) Nonlinear absorption and emission: In the late 1990s, Hell [18] developed the
methods to control the behaviours of fluorophores wherein all fluorophores are
switched off except the centremost fluorophores within the diffraction-limited
illumination volume of a laser scanning microscope, resulting in development of
stimulated emission depletion (STED) microscopy with typical lateral resolution as high as 30–50 nm (can reach 6 nm with nano-diamond). During the same
period, William Moerner demonstrated [19] that how certain mutants of green
fluorescent protein showed remarkable ‘blinking’ behaviour in their individual
fluorescence emission. After several rounds of blinking, these molecules would
go into stable dark state, and could be recovered by a short burst of UV light. This
idea had fascinated Betzig [20] to develop super-resolution microscope based
on blinking fluorophores and principle of localisation. The demonstration of
controlled on/off switching of fluorescent proteins had led to the discovery of
PALM with a resolution as high as 10 nm. It is important to note that all these
far-field super-resolution imaging techniques are all based on fluorescence as a
contrast mode.
In any fluorescence microscope, the high energy photon (from lamp or laser) excites
fluorophores in the specimen and the excited fluorophore subsequently emits its own
lower energy photon. The high-contrast imaging is achieved by using spectral filtering
of fluorescence from the excitation light. Imaging protein expression, localisation,
and activity in living cells is possible with the development of genetically encoded
fluorescent proteins [21, 22]. The high specificity of fluorescence light microscopy
facilitates the collection of spatial and functional information about bio-structures
using labelled molecules. In this way, the majority of light microscopy applications
in life sciences today use fluorescence as a dominating contrast mode.
However, the widely used fluorescence microscopy has certain shortcomings that
hinder further growth of the field. Some of these are: (1) Photo-bleaching: the fluorescent molecule on excitation over a period of time accumulates electrons in its excited
state that often leads to a chemical damage of fluorophore, resulting in loss of fluorescence, that is photo-bleaching. Photo-bleaching severely limits the time over which
a sample can be observed under microscope. (2) Photo-toxicity: this effect often
occurs as fluorescent molecules under intense illumination, which have a tendency
to generate reactive chemical species. Live cells are susceptible to them, resulting in
cell mortality. (3) Low photo-stability: for super-resolution imaging based on on/off
switching, typical fluorescent labels are not strong enough to undergo the switching
cycle for multiple times, thus limiting the length of total acquisition time. (4) Autofluorescence: This reduces contrast by forming an image background that even the
best excitation/emission filters could not completely get rid of. These shortcomings