Imaging of human meiotic chromosomes by scanning near-field optical microscopy (SNOM)

Hausmann, Michael; Liebe, Bodo; Perner, Birgit; Jerratsch, Martin; Greulich, Karl-Otto; Scherthan, Harry

doi:10.1016/s0968-4328(03)00021-0

“…The first two PCs explain 21 and 18 %, respectively, of the total variance. c The two loading plots of PC1 and PC2 reflect the variation in the Mie interference pattern contributed to each PC and how well each PC takes into account the variation in wavelength for the data points microscopy will enable spectroscopic analysis of substructures of a chromosome as small as 30 -50 nm [34,35]. Furthermore, by using Raman spectroscopy [36], chemical patterns, such as the DNA-to-protein ratios of segments, can be determined.…”

Section: Discussionmentioning

confidence: 99%

Hyperspectral backscatter imaging: a label-free approach to cytogenetics

Rebner

¹

,

Ostertag

²

,

Kessler

³

2016

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Current techniques for chromosome analysis need to be improved for rapid, economical identification of complex chromosomal defects by sensitive and selective visualisation. In this paper, we present a straightforward method for characterising unstained human metaphase chromosomes. Backscatter imaging in a dark-field setup combined with visible and short near-infrared spectroscopy is used to monitor morphological differences in the distribution of the chromosomal fine structure in human metaphase chromosomes. The reasons for the scattering centres in the fine structure are explained. Changes in the scattering centres during preparation of the metaphases are discussed. FDTD simulations are presented to substantiate the experimental findings. We show that local scattering features consisting of underlying spectral modulations of higher frequencies associated with a high variety of densely packed chromatin can be represented by their scatter profiles even on a sub-microscopic level. The result is independent of the chromosome preparation and structure size. This analytical method constitutes a rapid, cost-effective and label-free cytogenetic technique which can be used in a standard light microscope. Graphical abstract Hyperspectral backscatter imaging for label-free characterization.

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“…27) In this way, AFM examination of unstained chromosomal samples may provide us with new understandings of the structure/function relationships in chromosomes. 28) Furthermore, AFM and other new technologies such as scanning near-field optical microscopy 19,29,30) may allow more in-depth examinations of karyotypes and/or associations between chromosomal aberrations and disease in the future.…”

Section: Discussionmentioning

confidence: 99%

Atomic Force Microscopic Examination of Chromosomes Treated with Trypsin or Ethidium Bromide

Wu¹,

Cai²,

Long-qiu³

et al. 2006

CHEMICAL & PHARMACEUTICAL BULLETIN

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Recently, researchers have sought to better understand the relationship between chromosomal structure and function. Chromosomes, primarily composed of DNA and histone proteins, must replicate prior to cell division. During replication, errors can be introduced into the DNA via physical or chemical stressors, including heat, ultraviolet radiation, and other forms of radiation, heavy metal ions, drugs and organic reagents.1,2) However, it is also probable that chromosomal alterations might be introduced outside of replication, such as during the trypsinization or ethidium bromide (EB) staining processes 3) commonly used in visualization of chromosomes by optical microscopy. Changes of chromosome ultrastructure cannot be observed clearly under conventional optical microscopes due to technical difficulties such as diffraction limitations. However, the advent of atomic force microscopy (AFM) 4) has allowed researchers to investigate small alterations in chromosome structure and morphology.Previous studies have examined changes in chromosome structure caused by physicochemical treatments.5,6) Acetic acid, which is frequently used to remove cellular contaminants, was applied to barley chromosomes at concentrations of 15, 30 and 45%. AFM analysis revealed that the optimal concentration was 30%; at lower levels sufficiently clean chromosomes samples could not obtain and the chromosome surface structures could not be observed, and at higher levels the chromosome structures were significantly damaged, although the cellular contaminants were effectively removed. 7)Similar studies identified chromosomal damages induced by treatments with NaCl 8) and heavy ion radiation. 9)EB, a heterocyclic organic compound, can insert between base pairs, 10) resulting in increasing DNA rigidity and lengthening of DNA.11) Low doses of EB can induce DNA denaturation and can lead to cancer formation. 12) Thus, it is possible that EB staining of chromosome preparations may induce structural abnormalities 13) not present in the actual organism. Up to now, though there are some works about structural changes of chromosomes caused by physicochemical treatments, 3,5,6,[14][15][16] however, no previous work has used AFM to examine the effects of time-course of trypsin or different dose EB on chromosomal structures.In this article, we used AFM in the tapping mode to examine chromosomal damage induced by EB treatment and trypsin digestion in air. Our results showed that EB could profoundly damage human chromosomes, leading to breakages and separations in a dose-dependent fashion. We also found that trypsinization could obviously change the morphological features of chromosomes with increased digestion time; the optimal digestion time for AFM analysis appeared to be 10-20 s under our experimental conditions, but not to be over 40 s. In addition, our results indicate that AFM is a powerful tool for studying chromosomal damage, perhaps indicating that it will be applicable to further diagnostic applications. Together, our results indicate that common chr...

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“…Combinations with atomic force microscopy, laser tweezers, and immunolabeling, among others, extend the opportunities for application in life sciences from single fluorescently labeled molecules such as DNA or proteins up to whole cells and chromosomes (for examples, see [317][318][319][320][321][322]). SNOM is particularly suited to labeling cell surface membrane proteins, since the illumination depth is limited to tens of nanometers.…”

Section: Advanced Microscopic Techniques (Selection)mentioning

confidence: 99%

Instruments in Biotechnology and Medicine

Voss¹,

Feller²,

Beckmann³

2012

Handbook of Biophotonics

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The sections in this article are Photonic Instruments in Medicine Introduction Optical Window of Cells and Tissues Laser Light–Matter Interaction as a Function of Intensity Photochemical Treatment of Tissues Thermal Processes in Light–Matter Interaction Nonlinear Processes in Light–Matter Interaction (Not to Be Confused with Nonlinear Optics) Minimally Invasive Diagnostics – Endoscopy History Types and Components of Endoscopes Advantages and Disadvantages of Endoscopy Application Fields Combination of Standard White Light Endoscopic with Other Diagnostic Methods Some Trends in Endoscopy Noninvasive Diagnostics – Photoplethysmography Introduction Characterization of the PPG Signal Signal Acquisition Clinical Applications Summary Noninvasive Diagnostics – Ophthalmology Introduction Corneal Topography Perimetry Optical Biometry Retinal Imaging and Glaucoma Diagnostics Fluorescein Angiography Wavefront Analysis In Vitro Diagnostics – Microscopy Light Microscope Fluorescence Microscope New Fluorescence Microscope Techniques Confocal Microscopy Raman Microscopy In Vitro Diagnostics – Digital Slides and Virtual Microscopy In Vitro Diagnostics – Optical Methods in Clinical Analysis System Optical Spectroscopy of Blood and Urine Components Optical Spectroscopy of Tissues and Vessels Optical Spectroscopy of Agglutination and Precipitation – Nephelometry, Turbidimetry In Vitro Diagnostics – Blood Gas Pressure Measurements Optical Detection of p O 2 in Blood Optical Detection of p CO 2 in Blood Optical Detection of Further Monitoring Parameters in Intensive Care (Temperature, p H ) In Vitro Diagnostics – Photoacoustic Spectroscopy Therapy Introduction Laser Therapy in Ophthalmology – LASIK (Refraction Correction by Cornea Treatment) Photonic Instruments in Biotechnology Introduction Selected Analytical Methods Introduction Reflectometric Interference Spectroscopy ( RIf S ) Surface Plasmon Resonance ( SPR ) Fluorescence Methods Advanced Microscopic Techniques (Selection) Enzyme‐Linked Immunosorbent Assay ( ELISA ) Optical Biosensors, Biochips, Miniaturized Analytical Systems High‐Throughput Screening ( HTS ) Sample Preparation: Flow Injection Analysis Fermentation as a Central Process of Biotechnology General Aspects Fermentation Control Photobioreactors Optical Trap/Optical Tweezers Basic Scheme of Laser Tweezers and Micromanipulation Applications of Optical Tweezers

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Imaging of human meiotic chromosomes by scanning near-field optical microscopy (SNOM)

Cited by 16 publications

References 28 publications

Hyperspectral backscatter imaging: a label-free approach to cytogenetics

Hyperspectral backscatter imaging: a label-free approach to cytogenetics

Atomic Force Microscopic Examination of Chromosomes Treated with Trypsin or Ethidium Bromide

Instruments in Biotechnology and Medicine

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