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Optimum focusing of Gaussian beams with linear and radial polarization
No full textWhen a Gaussian beam is focused by a lens, the beam is truncated by the pupil, and energy is
lost. As the radius of the beam waist is increased, the size of the focal spot decreases, but
more energy is lost. Some papers have assumed particular truncation ratios, without
explaining why they made these particular choices. Electromagnetic focusing of planepolarized,
circularly-polarized, and radially-polarized Gaussian beams by an aplanatic lens of
high numerical aperture is considered. It is found that, unlike the paraxial case, for an
objective with a high numerical aperture there are optimum values for the truncation that
maximize either the intensity at the focus for a given input power, or the intensity at the focus
as compared with the side-lobe energy. For linearly-polarized illumination, the intensity at
the focus for a given input power is optimized for a specific Gaussian beam truncation ratio,
but increases monotonically with NA. The area of the focal spot (defined by the full-width at
half maximum, FWHM) is optimized for a specific NA, but decreases monotonically with
Gaussian beam truncation ratio. There is a global optimum (with respect to both truncation
ratio and NA) for the intensity at the focus as compared with the side lobe energy.
Various performance parameters can be calculated directly from the pupil function, without
the necessity for calculating the focal intensity distribution. These parameters include those
that are measures of the intensity at the focus, and the transverse gain GT , which is a measure
of the parabolic radius of the focal spot [1-4]. A new parameter P for comparing the purity
of the longitudinal field for different illumination beams is introduced. For radially-polarized
illumination, the transverse gain can be split into two parts, GTz and GTρ , originating from
the longitudinal and radial fields, respectively. The overall transverse gain is reduced by the
presence of the parasitic radial field. The polarization purity parameter is defined as
P = exp(GTρ /GTz ) , where 0 ≤ P ≤1 .
GT
and
P
are
found
to
be
greatest
for
illumination
by
what
we
have
called
an
axial
dipole
wave
(ADW)
[3],
and
also
increased
by
a
central
obscuration
of
the
pupil.
We
also
consider
radial
beam
generation
using
polarization
mode-‐conversion
of
a
linearly-‐polarized
Gaussian
beam.
The
concept
of
polarization
purity
is
also
applied
to
linearly-‐polarized
illumination
An application of surface roughness with CLSM: an algorithm to enhance the depth discrimination properties
No full textImage scanning microscopy with quadrant detectors
No full textThere is growing interest in techniques based on using detector arrays in confocal microscopy
[1]. Many different implementations have been proposed, and various different names given
to these techniques, one such name being image scanning microscopy [2]. The images from
the elements of the detector array can be combined optically [3], but more flexibility is
achieved by recording each image independently for subsequent processing. This can result
in a substantial improvement in overall image quality, with a combination of resolution
improvement and signal strength. The image is reconstructed by pixel reassignment, in which
the pixel value is reassigned to the appropriate object coordinates, which vary for different
points of the array. Alternatively, the data set consisting of images from different detector
pixels can be deconvolved using the known theoretical point spread function as a priori
information.
In order to maintain the optical sectioning property of the confocal microscope, the size of
the detector array should be limited to a region with a size of the order of the central lobe of
the focused Airy disc. Thus only a small number of detector elements are actually necessary,
a quadrant of four detector elements being a good desig
Comment on "Do evanescent waves really exist in free space?"
No full text10.1016/j.optcom.2006.05.046Optics Communications2662448-449OPCO
Can fluorescence and SHG data be enriched by Müller matrix signature
No full textThe most recent advances in optical microscopy are mainly focused towards superresolution,
using fluorescence stochastic/targeted read-out methods [1]. Since the demand is growing for
imaging thick biological specimens as cell aggregates (i.e., tumor spheroids), tissues/organs
(i.e. ligaments, meniscus) and small organisms (i.e. zebrafish), scattering represents a key
aspect in image analysis and reconstruction [2]. Additional information, if exploited
correctly, improves the accuracy of any measurements giving rise, in the specific case of
optical microscopy, to an increase in the bandwidth and hence resolution of the system [3].
So far, polarization properties of the incoming/outcoming light have been shown to be able to
provide further information about the sample [4, 5]. Here the attention is given to the
possibility of utilizing a Mueller matrix analysis of the signal in order to extract information
about optically active biological structures in the sample, with particular interest in chiral
objects [6-8]. Since Mueller matrix analysis is generally used to study polarization properties
in angular scattering, an ongoing question is: can fluorescence and SHG data be enriched by
Mueller matrix signature, too? As the possibility was demonstrated of obtaining
ultrastructural information about chromatin-DNA organization using circular intensity
differential light scattering [9], the Mueller matrix integrated approach could allow progress
towards label free imaging. A possible Mueller matrix polarimetry integrated architecture
will be outlined, based on photoelastic modulation (vs. Pockels cell) as a polarization
generator along the excitation pathway [9-13]. A classical electrodynamics model will be
reported about circular intensity differential scattering of chromatin-DNA in a label-free
perspective [14]. In addition, some preliminary raw data arising from SHG measurements
will be discussed
THE ROUTE FROM SUPER-RESOLUTION TO THE NOBEL PRIZE 2014
No full textSuper-resolution or super resolved fluorescence microscopy, as indicated in the Chemistry
Nobel Prize 2014 awarded to Eric Betzig, Stefan W. Hell and William Moerner, includes
those microscopy techniques that increase the resolving ability of a light microscope well
beyond the classical limits dictated by the diffraction barrier [1]. Since the end of the 19th
century Ernst Abbe (1873) and Lord Rayleigh (1896) clarified the reasons such a limit that
makes/made impossible to resolve two elements of a structure when they are closer to each
other than approximately 1⁄2 λ in the lateral (x,y) plane and ≈λ
along the axial direction (z).
So far, several methodologies have been developed over the past several years for superresolution
fluorescence microscopy including saturated structured-illumination microscopy
(SSIM), stimulated emission depletion microscopy (STED), photoactivated localization
microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and
stochastic optical reconstruction microscopy (STORM). Such a development had some
important “gregarios/sparring partners” in computational optical sectioning microscopy,
confocal and two-photon laser scanning microscopy, scanning near-field optical microscopy,
green fluorescent proteins advent and information communication approaches. The list is not
complete. Resolution improvements have been made with confocal and multiphoton
microscopy, as well with approaches like 4PI and I5M. However, in general, approaches
dealing with resolution improvements remained confined by Abbe’s and Rayleigh’s
prescriptions. We can also see the limit as set by concepts of information theory. I like to
mention the Toraldo di Francia approach related to super resolution [2] as starting point, and
to go across all those attempts and improvements predicted and implemented within the
scientific community focused on optical microscopy [3]. What is revolutionary today, in my
view, is the fact that there is theoretically no limit for capturing details by means of an optical
microscope and that, at the very same time, there is the possibility of tuning the spatial
resolution according to the scientific question posed.
In the style of Johannes Faber referred to the Galileo Galilei’s occhialino [4], one can modify
the sentence “microscopium nominare libuit” in “nanoscopium nominare libuit” for the superresolution
fluorescence microscope that has become a nanoscope
Near-IR Pump-probe microscopy for label-free superresolution imaging
No full textAt present, most optical microscopy techniques provide sub-diffraction scale imaging based on fluorescence as the underlying contrast mechanism. Fluorescence introduces certain limitations such as a reliance on labels, photo-bleaching and a reduction of penetration depth. Two-photon excitation microscopy, which utilizes near IR femtosecond lasers as a light source, overcomes these limitations. As a novel approach based on similar laser source, we implement the proposed absorption/saturation (pump-probe) microscopy method [1, 2]. Its principles are borrowed from pump-probe spectroscopy, where in order to investigate dynamic properties of an object two femtosecond laser beams are typically used. The first beam called the ‘pump’ modifies the carrier density inside the sample. This beam is followed by intensity changes during the transmission of a second (probe) beam, thereby creating a transient contrast. The method can be further improved by introducing a third ‘doughnut’ shaped beam, which arriving together along with the pump beam and saturate the induced transition within the periphery of the focal spot. As a result the transient contrast is generated only within the central area of sub-diffraction range dimension.
Our saturated transient absorption microscope (STAM) integrates this pump-probe spectroscope with a commercial Nikon microscope. The apparatus is based on a femtosecond laser coupled with an Optical Parametric Oscillator (OPO). The wavelengths of excitation and detection pulses can be tuned in accordance with experimental needs within the near IR region- a spectral range that is known to lie in the transparency window of biological tissues. By choosing specific wavelengths one can observe selected species and non-fluorescent markers. Our setup has the following capabilities: conventional confocal single and two-photon imaging both in reflection and in transmission, pump-probe imaging and saturated pump-probe. Combination of these techniques allows us to demonstrate explicit spatial and dynamic information for applications in cellular biophysics and nanochemistr
Background Rejection in Two-Photon Fluorescence Image Scanning Microscopy
No full textWe discuss the properties of signal strength and integrated intensity in two-photon excitation confocal microscopy and image scanning microscopy. The resolution, optical sectioning and background rejection are all improved over nonconfocal two-photon microscopy. Replacing the pinhole of confocal two-photon microscopy with a detector array increases the peak intensity of the point spread function. The outer pixels of a detector array give signals from defocused regions, and thus the processing of these, such as through subtraction, can further improve optical sectioning and background rejection.LE
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