Cut out or poke in—the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease

Schütze, Karin; Becker, Ingrid; Becker, Karl‐Friedrich; Thalhammer, Stefan; Stark, Robert W.; Heckl, Wolfgang M.; Böhm, Malte; Pösl, H.

doi:10.1016/s1050-3862(96)00169-6

Cited by 59 publications

(34 citation statements)

References 43 publications

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“…A sample set of this data is shown in Figure 6b. Conservation of mass in this system provides the following relationship between the external fluid velocity V ∞ (r, t) and the bubble dynamics as: (1) Conservation of momentum was then applied to obtain the following expression for the wall shear stress τ w (r, t) generated by the cavitation bubble expansion [20]: (2) where ρ and ν are is the density and kinematic viscosity of the fluid medium [20], Equation (2) is valid at any radial position r and time 0 ≤ t ≤ t′ where t′ is the time of arrival of the bubble wall at position r.…”

Section: Role Of Cavitation Bubble Generated Shear Stress On Cell Lysmentioning

confidence: 99%

“…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 catapulting [2][3][4][5], cell collection, expansion, and purification [6][7][8], cellular microsurgery [9][10][11], 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 cells [16][17][18][19][20][21][22], 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.…”

Section: Introductionmentioning

confidence: 99%

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Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Hellman

Rau

Yoon

et al. 2008

Journal of Biophotonics

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Cell lysis and molecular delivery in confluent monolayers of PtK 2 cells are achieved by the delivery of 6 ns, λ = 532 nm laser pulses via a 40×, 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 (3kDa) 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 τ w, max > 190 ± 20 kPa are immediately lysed while cells exposed to τ w, max > 18 ± 2kPa are necrotic and subsequently detach. Cells exposed to τ w, max in the range 8-18 kPa are viable and successfully optoporated with 3kDa Dextran molecules. Cells exposed to τ 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.

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Section: Role Of Cavitation Bubble Generated Shear Stress On Cell Lysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Hellman

Rau

Yoon

et al. 2008

Journal of Biophotonics

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“…To reliably prepare mRNAs rapidly from a single cell, there appeared to be two possibilities: (1) aspiration of the intracellular contents with a patch pipette (Monyer and Lambolez, 1995;Surmeier et al, 1996;Baro et al, 1997;Liss et al, 2001;Tsuzuki et al, 2001;Liss, 2002;Song, 2002); and (2) isolation of the cell's soma by laser microdissection (Schütze et al, 1997;Fink et al, 1998;Mawrin et al, 2003). As with any model system or experimental procedure, there are various 'pluses and minuses' that must be taken into account before proceeding.…”

Section: Introductionmentioning

confidence: 99%

Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR

Wagatsuma

Sadamoto

Kitahashi

et al. 2005

Journal of Experimental Biology

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SUMMARY Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. However, there are no accurate, rapid and appropriate methods to determine the exact copy numbers of particular mRNAs in a single cell. We therefore developed a procedure for isolating a single, identifiable cell and determining the exact copy numbers of mRNAs within it. We first isolated the cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior brought about by associative learning of feeding behavior. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs). These transcription factors play an important role in memory formation across animal species. The protocol uses two techniques in concert with each other: a technique for isolating a single neuron with newly developed micromanipulators coupled to an assay of mRNAs by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The molecular assay determined the mRNA copy numbers, each of which was compared with a standard curve prepared from cDNA solutions corresponding to the serially diluted solutions of Lymnaea CREB mRNA. The standard curves were linear within a range of 10 to 105 copies, and the intra-assay variation was within 15%. Each neuron removed from the ganglia was punctured to extract the total RNA directly and was used for the assay without further purification. Using this two-step procedure, we found that the mRNA copy number of CREB repressor (CREB2) was 30–240 in a single cerebral giant cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay (<25). These results suggest that the CREB cascade is regulated by an excess amount of CREB2 in the cerebral giant cells. Our procedure is the only quantitative analysis for elucidation of the dynamics of gene transcription in a single cell.

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“…They are the infrared (IR) capture systems [331,332] and the ultraviolet (UV) cutting systems [333][334][335][336][337][338]. All commercially available LCM systems are based on one of these two platforms [326].…”

Section: Two Types Of Lcmmentioning

confidence: 99%

“…Then the film is lifted and the cells together with the film are transferred to a micro-centrifuge tube containing buffer solutions for the isolation of DNA or RNA [328]. In 1996, Schütze et al developed the second platform with the UV laser microbeam microdissection system [333]. In this system, the cells or regions of interest are cut by a highly focused laser beam.…”

Section: Two Types Of Lcmmentioning

confidence: 99%

Technique and method development for intervertebral disc (IVD) research

Lee¹,

李芷恩²

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Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding.Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry.In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process.However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.(484 words)

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Cut out or poke in—the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease

Cited by 59 publications

References 43 publications

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR

Technique and method development for intervertebral disc (IVD) research

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