It has been shown recently that samples containing hemoglobin exhibit auto fluorescence upon two photon excitation. This property enables label free imaging of erythrocytes and other hemoglobin features with minimal and simple sample preparation. We utilized this effect for imaging of human and animal erythrocytes in various physiological condition, as well as empty erythrocytes' membranes that are used as potential vehicles for targeted drug delivery in order to map residual hemoglobin after the process of gradual hypotonic hemolysis (click here for reference).
The fluorescence from hemoglobin sample upon two photon excitation exhibits some peculiarities (e.g. increase of the fluorescence intensity when the incident laser intensity is increased, exactly the opostie of photobelaching) and its origin is not known [1, 2]. This property of interaction between hemoglobin and ultrashort laser pulses, along with other properties of erythrocites in various pathophysiological conditions, new microscopic technique build up (Fluorescence correlation spectroscopy link na nase radove sa FCS) will be thoroughly studied in the HEMMAGINERO project within PROMIS program of Science Fund of Republic of Serbia.
Hemoglobin properties will be examined also in THz region using the radiation form the free lectron laser. This studies will be performed through the bilateral project with THz beam line group at FLASH, DESY, Hamburg Germany. Also, super resolution methods and hyper spectral imaging will be applied to address the issues in more thorough way. This will be accomplished through the bilateral project with Laboratory of Biophysics, Jožef Štefan Institute, Ljubljana, Slovenia.
Imaging and micro processing of chitinous structures for documents security and entomology
Chitin, polysaccharide, the main building compound in arthropod's world, exhibits strong autofluorescence
when excited in blue (one photon excitation) or near IR (two photon excitation) region.
Therefore, structures made of chitin are suitable for label free imaging which is applicable for
assessing various problems in biology, biophotonics, document security etc.
Entomology: Due to high penetration depth of NLSM it is possible to access the deeper structures
and to image them without destroying the sample. This property was utilized in entomological i.e. taxonomical
studies for rare species. In addition, we showed that chitin can exhibit weak SHG signal.
Documents protection: Have you ever touched the butterfly wings? Have you seen the powder on your fingertips?
The powder you saw consists of number of scales that cover the wing of a butterfly and those scales are made out of chitin, too.
In addition, the scales are patterned in a "diffraction grating like" style, which makes them even more interesting as biophotonical structures.
Colon tissue imaging and early stage cancer diagnostic
Polarization resolved SHG (PHSG) imaging is the golden standard
for collagen fibers organization studies nowadays. From the other hand, (dis)organization of collagen fibers
is closely related to the degree of cancer of certain tissues. We applied PSHG imaging for collagen fiber organization analysis
in malignant tissues of human colon. In addition we examined the "healthy" tissue that is at certain distance from the malignant one
in order to predict if it is suspicious to the cancer or not.
The SHG anisotropy could be used to quantify alignment of collagen molecules inside
fibers. The anisotropy parameter ß was calculated by:
where Ipar and Iorth represented SHG intensity detected when the analyzing polarizer is
oriented parallel (Ipar) and orthogonal (Iorth) to the laser polarization [1, 2]. Values of β range from 0 to 1, where 0 represents completely random and 1 completely aligned collagen molecules inside fibers.
Polarization-resolved SHG imaging as a fast screening method for collagen
alterations during aging
Skin aging is a complex phenomenon that includes chronological and photoaging, which
are both oxidative damage-related processes. Previous study on rat skin showed that
cumulative oxidative pressure during aging induces profound structural and ultrastructural
alterations in rat skin epidermis and dermis . Here, we focus on underlying structural alterations of the dermis components during chronological aging.
We used second harmonic generation (SHG) and two-photon excited fluorescence (TPEF)
on 5 µm thick skin paraffin sections from 15-day-, 1-month- and 21-month-old rats,
respectively, to analyze collagen alterations, in comparison to conventional light and
electron microscopy methods. Obtained results show that polarization-resolved SHG (PSHG)
images can detect collagen fiber alterations in line with chronological aging and that this
method is consistent with light and electron microscopy. Moreover, the ß coefficient
calculated from PSHG images points out that delicate alterations lead to a more ordered
structure of collagen molecules due to oxidative damage. The results of this study also open
the possibility of successfully applying this fast and label-free method to previously fixed
Design of novel fluorescent probes for biomedical imaging based on up-converting nanoparticles
Fluorescent probes commonly found nowadays are dye molecules that are bind for the specific protein and enable imaging.
In order to overcome some drawbacks of standard fluorescent dye molecules (photobelaching, wither one or two photon excitation,
nonspecificity, low quantum yield, unsuitable spectral properties etc), novel fluorescent probes based on up converter nanoparticles
have been developed. We utilized NLSM to test and analyize the properties of novel probes developed by our colaborators.
Dental tissues, dentin and enamel, can be imaged by TPEF and SHG microscopy. We have combined information obtained by NLSM and Brlouin spectroscopy for the mechanical properties studies of dental tissue.
Cell surgery and electrophysiology
TPEF: excitation 730 nm, detection 400-700 nm
THG: excitation 1040 nm, detection 347 nm
Pattern inscription: cutting and cell micro surgery
In addition to the imaging, our microscopic system is able to process the samples
and inscribe arbitrary patterns by controllable delivery of the laser energy to the focal spot
(laser intensity, dwell time, writing speed). This level of control enables the cell surgery by the laser beam.
We have applied the cell surgery mechanism to enable electrophysiological studies, i.e. patch clamping, on the hifae of
Phycomices Bleackensianus. Namely, in addition the plasma membrane that is to be studied by electrophyiology,
hyphae have the chitinous cell wall around the membrane, that prevents direct access to the membrane by patch clam pipette.
The aim of this study is to make an laser incision in the cell wall and to open the direc access for patch clamping.
In addition to the TPEF imaging, the transmission arm of the microscope that will enable label free THG imaging is planned
to be built up through the HEMMAGINERO project within PROMIS program of Science Fund of Republic of Serbia.
The design and equipment specification is already done, and some preliminary results are achieved with replaceable equipment.
Second harmonic generation in doped polymeric carbon monoxide
Michael G. Pravica, Department of Physics and Astronomy, University of Nevada
Las Vegas (UNLV), USA
Second harmonic generation (SHG) experiments were conducted on samples of a
novel material: doped polymeric CO (dp-CO) that were synthesized via synchrotron X-ray irradiation of SrC2O4 and high pressure. The goal of the study was to ascertain if the novel material is rugged enough to handle high levels of radiation to enable its possible use as future sensors or optoelectronic devices. Three samples were tested.
Two of the smaller samples that were synthesized in a diamond anvil cell (DAC) and
recovered at ambient conditions rapidly decomposed/degraded in the presence of the
strong 3.6×109 mW/cm2 intensity optical beam. This lends credence that this novel
material (when synthesized under select conditions) may offer utility as a rugged
radiation hardened and easy to manufacture nonlinear optical device.