Fig 4 - uploaded by Vincent Patrick Wallace
Content may be subject to copyright.
( A ) A mitotic cell with chromosomes clearly visible aligned at the metaphase plate. ( B ) The same cell with the chromosomes in the center of the metaphase plate exhibiting two-photon fluorescence during exposure to the 1.06- m-wavelength laser beam focused through the ϫ 63 microscope objective. Cells were exposed to ethidium monoazide bromide at 10 m ͞ 1 ml for 24 h. (Bar ϭ 15 microns.)
Source publication
Multiphoton-targeted photochemistry was used to selectively inactivate the expression of genes in vertebrate cells. A membrane permeable DNA-associating vital dye, ethidium bromide monoacetate (visible wavelength single photon absorption peak at 530 nm) was used to photosensitize chromosomes in dividing cells. A 100-ps infrared laser beam operating...
Context in source publication
Context 1
... two-photon excitation capability of the picosecond laser microscope was readily demonstrated in PTK 2 cells treated with the ethidium monoazide bromide vital dye. The 1.06-m wave- length laser beam was focused onto individual chromosomes of dividing live cells. Bright red-orange fluorescence was detected visually (Fig. 4 A and B) and was subsequently collected over 5-s periods by using an Acton Research (Acton, MA) SpectraPro- 150 spectrometer coupled to a cooled CCD detector (Princeton Instruments TECCD-576E) mounted on the laser microscope. A loglog plot (slope 1.95) of fluorescence intensity versus laser power was derived by stepwise attenuation of the laser ...
Citations
... The study of MP absorption can yield new information about the basic interactions within and between molecules. Due to the fact that spatial resolution of two-photon (2-P) absorption can produce subcellular destructive events (Berns, Wang et al. 2000), MP ...
... Meanwhile, several different types of laser systems are frequently used to generate localized DNA damage, but a comparison between the obtained data is often difficult as the results depend strongly on the wavelength (Mohanty et al. 2002;Kong et al. 2009) and the energy (Rogakou et al. 1999;Kong et al. 2009) of the laser system used. With the application of multiphoton laser systems (Meldrum et al. 2003;Mari et al. 2006), the duration of laser pulses also has a major impact on the damage outcome (Berns et al. 2000;Kong et al. 2009). To unify this diversity of impact factors, it would be useful to find a single parameter correlated with the amount of laser-induced DNA damage. ...
... Meanwhile, several different types of laser systems are frequently used to generate localized DNA damage, but a comparison between the obtained data is often difficult as the results depend strongly on the wavelength (19,20) and the energy (6,20) of the laser system used. With the application of multiphoton laser systems (2,21), the duration of laser pulses also has a major impact on the damage outcome (20,22). To unify this diversity of impact factors, it would be useful to find a single parameter correlated with the amount of laser-induced DNA damage. ...
The induction of localized DNA damage within a discrete nuclear volume is an important tool in DNA repair studies. Both charged particle irradiation and laser microirradiation (LMI) systems allow for such a localized damage induction, but the results obtained are difficult to compare, as the delivered laser dose cannot be measured directly. Therefore, we revisited the idea of a biological dosimetry based on the microscopic evaluation of irradiation-induced Replication Protein A (RPA) foci numbers. Considering that local dose deposition is characteristic for both LMI and charged particles, we took advantage of the defined dosimetry of particle irradiation to estimate the locally applied laser dose equivalent. Within the irradiated nuclear sub-volumes, the doses were in the range of several hundreds of Gray. However, local dose estimation is limited by the saturation of the RPA foci numbers with increasing particle doses. Even high-resolution 4Pi microscopy did not abrogate saturation as it was not able to resolve single lesions within individual RPA foci. Nevertheless, 4Pi microscopy revealed multiple and distinct 53BP1- and gamma H2AX-stained substructures within the lesion flanking chromatin domains. Monitoring the local recruitment of the telomere repeat-binding factors TRF1 and TRF2 showed that both proteins accumulated at damage sites after UVA-LMI but not after densely ionizing charged particle irradiation. Hence, our results indicate that the local dose delivered by UVA-LMI is extremely high and cannot be accurately translated into an equivalent ionizing radiation dose, despite the sophisticated techniques used in this study.
... This effect was observed experimentally in the early 1960's [2]. Since then 2PA has become a valuable practical tool especially in high resolution microscopy [3][4][5][6][7][8][9][10][11][12], and also in many other applications, such as ultrafast optical power limiting [13][14][15][16][17][18][19][20][21][22][23][24][25][26], deep tissue photodynamic therapy (PDT) [27][28][29][30][31][32][33][34][35][36][37][38], volumetric 3D optical memory , 3D microfabrication [60][61][62][63][64][65][66][67][68][69], and ultrafast pulse characterization [70][71][72][73][74]. Shortly after the first experimental observation, 2PA also was engaged as a spectroscopic tool, suitable to study transitions to states whose one-photon transitions are prohibited by parity selection rules [75][76][77][78][79][80][81][82][83]. ...
This dissertation explores quantitative two-photon absorption spectroscopy to relate molecular structure with optical properties of organic chromophores. The dissertation describes an advanced fluorescence-based technique for reliable measurements of the two-photon spectra and cross sections. To facilitate the measurements it establishes a set of reference compounds measured with a 15% absolute accuracy covering a broad range of excitation and fluorescence wavelengths. The dissertation shows that in many cases the few-essential-levels model can be successfully applied for the description and interpretation of two-photon absorption spectra and cross sections, at least for the low-energy transitions. The dissertation presents examples of applications of two-photon absorption for volumetric optical storage and cancer tumor detection. It describes the basic principles of the two-photon absorption-based optical memory and limitations imposed on two-photon sensitivity of photochromic materials by a necessity of fast access to the data. It also proposes a novel technique for sensitive detection of cancer cells by using two-photon excitation of near-IR fluorescence of a commercial dye and discusses the mechanisms responsible for differentiation between the normal and the cancer cells. The methods described in this dissertation can be applied to understanding the relations between structure and two-photon absorption strength of individual transitions of organic and biological chromophores, which can be used for design of new materials, maximally adapted for particular applications.
... There is no known molecular basis for these nucleolar visitations, and no obvious reason, notwithstanding speculation (e.g., Pederson and Politz, 2000 ), why SRP assembly or U2 and U6 RNA modifi cation should occur in the nucleolus. To learn if the nucleolus is essential for these latter functions, one would need new experimental approaches such as using the X. laevis embryo homozygous for the anucleolate mutation ( Elsdale et al., 1958 ) or cells in which the nucleoli are ablated by hyper-focused, thermally minimal laser irradiation ( Berns et al., 2000 ). ...
The life of the nucleolus has proven to be more colorful and multifaceted than had been envisioned a decade ago. A large number of proteins found in this subnuclear compartment have no identifiable tie either to the ribosome biosynthetic pathway or to the other newly established activities occurring within the nucleolus. The questions of how and why these proteins end up in this subnuclear compartment remain unanswered and are the focus of intense current interest. This review discusses our thoughts on the discovery of nonribosomal proteins in the nucleolus.
... Two-photon scanning laser microbeams have revolutionized the field of functional biological imaging (11) and manipulation (12,13). The focal point specificity of the technique provides the capability to image objects at the focal plane with little or no collateral excitation and can be used to selectively inactivate genes (13), and reduced scattering cross section and absorption in the tissue ''optical window'' at 700-1000 nm provides deeper penetration. ...
... Two-photon scanning laser microbeams have revolutionized the field of functional biological imaging (11) and manipulation (12,13). The focal point specificity of the technique provides the capability to image objects at the focal plane with little or no collateral excitation and can be used to selectively inactivate genes (13), and reduced scattering cross section and absorption in the tissue ''optical window'' at 700-1000 nm provides deeper penetration. Indeed, single-photon activation of ChR2 was recently coupled with two-photon calcium (Ca) imaging for visualization of activation processes in cells (14). ...
We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.
... Pulsed laser microbeams offer the ability to deposit energy with high spatial precision to produce controllable perturbations to cellular systems. As a result, there is an increasing interest to use pulsed laser microbeams for precise cellular manipulation, including laserinduced cell lysis [1], cell microdissection and catapulting2345, cell collection, expansion, and purification678, cellular microsurgery91011, and cell membrane permeabilization for the delivery of membrane-impermeant molecules into cells [12 15], The processes of laser-induced optoinjection and optoporation offer the ability to load cells with a variety of biomolecules on short time scales (milliseconds to seconds) through optically produced cell membrane permeabilization [12, 14, 15], Despite the innovative utilization of laser microbeams in cell biology and biotechnology, only recently have studies provided insight regarding the mechanisms that mediate the interactions of highly focused pulsed laser beams with cells16171819202122, A better understanding of these processes will prove critical to the continued development of laser microbeams for both research and practical applications. In previous studies, we provided a detailed characterization of the physics involved in the interaction of highly-focused nanosecond laser microbeams with cells [19, 20], However, it is important to relate these physical effects to the biological response of the cells. ...
Cell lysis and molecular delivery in confluent monolayers of PtK(2) cells are achieved by the delivery of 6 ns, lambda = 532 nm laser pulses via a 40x, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3 kDa) FITC-conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time-resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam-induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses tau(w, max) > 190 +/- 20 kPa are immediately lysed while cells exposed to tau(w, max) > 18 +/- 2 kPa are necrotic and subsequently detach. Cells exposed to tau(w, max) in the range 8-18 kPa are viable and successfully optoporated with 3 kDa Dextran molecules. Cells exposed to tau(w, max) < 8 +/- 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam-induced shear stresses and subsequent cellular outcome.
... EMA selectively labels cells with damaged nuclear membranes. It binds to the DNA of dead nuclei covalently, thereby preventing dye leakage after fixation (Berns et al., 2000). ...
The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage.
... However, a comparison of the irradiance thresholds for photodamage with the corresponding free electron densities in figure 21 shows that the formation of low-densitiy plasmas may contribute considerably to the observed damage or even dominate its creation, especially in non-stained targets. An exception is the work by Berns et al. (2000) who achieved gene manipulation by exposing chromosomes that were sensitized with a dye absorbing at 530 nm wavelength to 100-ps pulses of 1064 nm wavelength. The peak irradiance used in these experiments was ≈ 10 7 W/cm 2 , far below the threshold for plasma formation, and it is quite clear that in this case the laser effects relied on two-photon-induced photochemical changes without the involvement of free electrons. ...
Femtosecond laser pulses enable the creation of spatially extremely confined chemical, thermal, and mechanical effects in biological media and other transparent materials via free-electron generation through nonlinear absorption. The principle mechanisms of femtosecond laser interaction with biomaterials are not only relevant for nanosurgery with tightly focused laser pulses but also for applications such as intrastromal corneal refractive surgery or presbyopia treatment where the laser pulses are focused at smaller numerical apertures. A significant advancement has been made in models of optical breakdown in biomaterials from this project, specifically from the understanding of specific absorption properties of the biomolecules or strains contained in the aqueous medium.
... Why would one need 24-h access to the laser microscope? In our case, this is needed to perform delicate subcellular manipulations when individual cells either are at a particular stage in the cell cycle, or need to be manipulated at some point following a previous manipulation ( Berns et al., 2000;McNeill and Berns, 1981). Additionally, the needs to follow, to further manipulate, and to gather optical images of either individual or multiple cells after laser manipulation necessitate the development of software-hardware interfaces as well as machine-learning algorithms that allow decisionbased operations (Berns and Berns, 1982;Huang and Murphy, 2004;Price et al., 2002). ...
We have engineered a robotic laser ablation and tweezers microscope that can be operated via the internet using most internet accessible devices, including laptops, desktop computers, and personal data assistants (PDAs). The system affords individual investigators the ability to conduct micromanipulation experiments (cell surgery or trapping) from remote locations (i.e., between the US and Australia). This system greatly expands the availability of complex and expensive research technologies via investigator-networking over the internet. It serves as a model for other "internet-friendly" technologies leading to large scale networking and data-sharing between investigators, groups, and institutions on a global scale. The system offers three unique features: (1) the freedom to operate the system from any internet-capable computer, (2) the ability to image, ablate, and/or trap cells and their organelles by "remote-control," and (3) the security and convenience of controlling the system in the laboratory on the user's own personal computer and not on the host machine. Four "proof of principle" experiments were conducted: (1) precise control of microscope movement and live cell visualization, (2) subcellular microsurgery on the microtubule organizing center of live cells viewed under phase contrast and fluorescence microscopy, (3) precise targeting of multiple sites within single red blood cells, and (4) optical trapping of 10 microm diameter polystyrene microspheres.
