Continuous Wave Two-Photon Scanning Near-Field Optical Microscopy

Kirsch, Achim K.; Subramaniam, Vinod; Striker, G.; Schnetter, Christoph M.; Arndt‐Jovin, Donna J.; Jovin, Thomas M.

doi:10.1016/s0006-3495(98)74070-6

Cited by 27 publications

(31 citation statements)

References 30 publications

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“…Using 4 Pi two‐photon microscopy, axial resolutions of about 100 nm have been demonstrated (Hell & Stelzer, 1992; Hell et al ., 1997; Schrader et al ., 1998a,b). Recently, elegant high‐resolution two‐photon near‐field scanning microscopy has been realized (Hell et al ., 1998; Kirsch et al ., 1998; Jenei et al ., 1999). Furthermore, single molecule detection with two‐photon fluorescence correlation spectroscopy has been demonstrated (So et al ., 1998; Schwille et al ., 1999).…”

Section: Experimental Set‐up and Features Of Multiphoton Microscopesmentioning

confidence: 99%

Multiphoton microscopy in life sciences

König

2000

Journal of Microscopy

1,180

842

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SummaryNear infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm 22 to GW cm 22 , which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depthresolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells.Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a onephoton or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm 22 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible.

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Section: Experimental Set‐up and Features Of Multiphoton Microscopesmentioning

confidence: 99%

Multiphoton microscopy in life sciences

König

2000

Journal of Microscopy

1,180

842

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“…Therefore it would be difficult to distinguish between the contribution of stress induced by cadmium or by phototoxicity. Furthermore, high‐energy UV light is usually scattered by biological tissue, impeding high image quality (Kirsch et al ., 1998). The SAC8.5 CCD camera showed a low and stable dark noise with respect to the fluorescence signal allowing good quality images.…”

Section: Discussionmentioning

confidence: 99%

A highly reliable and budget‐friendly Peltier‐cooled camera for biological fluorescence imaging microscopy

Jolling

vandeVen

Eynden

et al. 2007

Journal of Microscopy

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SummaryThe SAC8.5, a low-cost Peltier-cooled black and white 8-bit CCD camera for astronomy, was evaluated for its use in imaging microscopy. Two camera-microscope configurations were used: an epifluorescence microscope (Nikon Eclipse TE2000-U) and a bottom port laser scanning confocal microscope system (Zeiss LSCM 510 META). Main advantages of the CCD camera over the currently used photomultiplier detection in the scanning setup are fast image capturing, stable background, an improved signalto-noise ratio and good linearity. Based on DAPI-labelled Chinese Hamster Ovarian cells, the signal-to-noise ratio was estimated to be 4 times higher with respect to the currently used confocal photomultiplier detector. A linear relationship between the fluorescence signal and the FITCinulin concentrations ranging from 0.05 to 1.8 mg mL −1 could be established. With the SAC8.5 CCD camera and using DAPI, calcein-AM and propidium iodide we could also distinguish between viable, apoptotic and necrotic cells: exposure to CdCl 2 caused necrosis in A6 cells. Additional examples include the observation of wire-like mitochondrial networks in Mito Tracker Green-loaded Madin-Darby canine kidney cells. Furthermore, it is straightforward to interface the SAC8.5 with automated shutters to prevent rapid fluorophore photobleaching via easy to use ASTROVIDEO software. In this study, we demonstrate that the SAC8.5 black and white CCD camera is an easy-to-implement and cost-conscious addition to quantitative fluorescence microfluorimetry on living tissues and is suitable for teaching laboratories.Correspondence to: Koen Jolling. Tel: 0032 (0)11 268532; fax: 0032 (0) 11 16 8599;

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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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Continuous Wave Two-Photon Scanning Near-Field Optical Microscopy

Cited by 27 publications

References 30 publications

Multiphoton microscopy in life sciences

Multiphoton microscopy in life sciences

A highly reliable and budget‐friendly Peltier‐cooled camera for biological fluorescence imaging microscopy

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

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