Hasil untuk "Microscopy"

G. Gordon, G. Berry, X. Liang et al.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.

K. Maslov, Hao F. Zhang, Song Hu et al.

W. Choi, C. Fang-Yen, K. Badizadegan et al.

W. Chao, W. Chao, B. Harteneck et al.

S. Cuenot, C. Fretigny, S. Demoustier‐Champagne et al.

B. Fultz, J. Howe

W. E. Moernera, D. P. Fromm

B. Vakoc, Ryan M Lanning, J. Tyrrell et al.

T. Kuznetsova, M. Starodubtseva, N. Yegorenkov et al.

David N. Breslauer, R. Maamari, N. Switz et al.

Light microscopy provides a simple, cost-effective, and vital method for the diagnosis and screening of hematologic and infectious diseases. In many regions of the world, however, the required equipment is either unavailable or insufficiently portable, and operators may not possess adequate training to make full use of the images obtained. Counterintuitively, these same regions are often well served by mobile phone networks, suggesting the possibility of leveraging portable, camera-enabled mobile phones for diagnostic imaging and telemedicine. Toward this end we have built a mobile phone-mounted light microscope and demonstrated its potential for clinical use by imaging P. falciparum-infected and sickle red blood cells in brightfield and M. tuberculosis-infected sputum samples in fluorescence with LED excitation. In all cases resolution exceeded that necessary to detect blood cell and microorganism morphology, and with the tuberculosis samples we took further advantage of the digitized images to demonstrate automated bacillus counting via image analysis software. We expect such a telemedicine system for global healthcare via mobile phone – offering inexpensive brightfield and fluorescence microscopy integrated with automated image analysis – to provide an important tool for disease diagnosis and screening, particularly in the developing world and rural areas where laboratory facilities are scarce but mobile phone infrastructure is extensive.

G. Popescu, Takahiro Ikeda, R. Dasari et al.

N. Wilson, P. Pandey, R. Beanland et al.

J. Waters

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

F. Subach, G. Patterson, S. Manley et al.

J. Huisken, D. Stainier

L. Gross, Fabian Mohn, N. Moll et al.

Alex Terrasson, Lars Madsen, Joel Grim et al.

Squeezed states of light enable enhanced measurement precision by reducing noise below the standard quantum limit. A key application of squeezed light is nonlinear microscopy, where state-of-the-art performance is limited by photodamage and quantum-limited noise. Such microscopes require bright, pulsed light for optimal operation, yet generating and detecting bright pulsed squeezing at high levels remains challenging. In this work, we present an efficient technique to generate high levels of bright picosecond pulsed squeezed light using a $χ^2$ optical parametric amplification process in a waveguide. We measure $-3.2~\mathrm{dB}$ of bright squeezing with optical power compatible with nonlinear microscopy, as well as $-3.6~\mathrm{dB}$ of vacuum squeezing. Corrected for losses, these squeezing levels correspond to $-15.4^{+2.7}_{-8.7}~\mathrm{dB}$ of squeezing generated in the waveguide. The measured level of bright amplitude pulsed squeezing is to our knowledge the highest reported to date, and will contribute to the broader adoption of quantum-enhanced nonlinear microscopy in biological studies.

Macarena Morillo-Huesca, Ignacio G. López-Cepero, Ryan Conesa-Bakkali et al.

Abstract Background Tumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear. Methods High-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients were used to identify novel, biologically relevant AEP targets. Immunoblotting and qPCR analyses were used to quantify protein and mRNA levels. Flow cytometry, confocal microscopy, chemical inhibitors, siRNA- and shRNA-silencing and DNA repair assays were used as functional assays. In-silico analyses using the TCGA BC dataset and immunofluorescence assays in an independent cohort of invasive ductal (ID) BC patients were used to validate the clinical relevance of our findings. Results Here we showed a dual role for AEP in genomic stability and radiotherapy resistance in BC patients by suppressing ATR and PPP1R10 levels. Reduced ATR and PPP1R10 levels were found in BC patients expressing high AEP levels and correlated with worst prognosis. Mechanistically, AEP suppresses ATR levels, reducing DNA damage-induced cell death, and PPP1R10 levels, promoting Chek1/P53 cell cycle checkpoint activation, allowing BC cells to efficiently repair DNA. Functional studies revealed AEP-deficiency results in genomic instability, increased DNA damage signaling, reduced Chek1/P53 activation, impaired DNA repair and cell death, with phosphatase inhibitors restoring the DNA damage response in AEP-deficient BC cells. Furthermore, AEP inhibition sensitized BC cells to the chemotherapeutic reagents cisplatin and etoposide. Immunofluorescence assays in an independent cohort of IDBC patients showed increased AEP levels in ductal cells. These analyses showed that higher AEP levels in radioresistant IDBC patients resulted in ATR nuclear eviction, revealing AEPhigh/ATRlow protein levels as an efficient predictive biomarker for the stratification of radioresistant patients. Conclusion The newly identified AEP/ATR/PPP1R10 axis plays a dual role in genomic stability and radiotherapy resistance in BC. Our work provides new clues to the underlying mechanisms of tumor resistance and strong evidence validating the AEP/ATR axis as a novel predictive biomarker and therapeutic target for the stratification and treatment of radioresistant BC patients.

Timothy J. Pegg, Daniel K. Gladish, Robert L. Baker

Flooding can cause root hypoxia and can lead to significant agricultural losses. Therefore, understanding plant adaptations to flooding, including root aerenchyma development, is one important avenue for insuring future global food security. We investigated cell wall modifications during root aerenchyma formation in response to the prolonged 0–48 h flooding of <i>Phaseolus coccineus</i>, <i>Pisum sativum</i>, and <i>Cicer arietinum</i> seedlings. Using transmission electron microscopy, toluidine blue O (TBO) staining, and immunolabeling with antibodies targeting de-methyl-esterified homogalacturonan (DMEH), partially DMEH, and methyl-esterified homogalacturonan (MEH), we examined changes in cell wall composition. Transmission electron microscopy and TBO staining revealed degradation of cell walls and middle lamella, with accumulation of DMEH near flooding-induced aerenchyma cavities. Immunolabeling indicated increased DMEH epitope availability in flooded roots, suggesting a role in cell wall remodeling. Enzyme pretreatments, used to “unmask” homogalacturonan by removing cellulose and hemicellulose, revealed that specific forms of homogalacturonan, particularly DMEH complexed with calcium and MEH, are masked by these cell wall components. The study highlights the complex interplay of pectin, cellulose, and hemicellulose in cell wall degradation during aerenchyma development, providing insights into legume flooding stress responses.

Shupei Lin, Nanfang Jiao, Yevhenii Shaidiuk et al.

We introduce and experimentally implement Fourier-plane phase synchronization for optical microscopy, and demonstrate its performance with interferometric scattering microscopy. By combining a photothermal phase plate and laser beam scanning, we realize a synchronized phase for all scattering components on the Fourier plane of high numerical-aperture microscopes, where the evanescent waves and optical aberration normally produce highly inhomogeneous phase distributions. We achieve an almost perfect point spread function, exhibiting a tighter focus with 50\% enhancement of the signal and ideal circular symmetry. Particularly, by synchronizing the phase to $π/2$, we demonstrate the background speckles exhibit an anti-symmetric dependence on axial defocus, enabling the effective suppression of the speckles via defocus integration and thus the detection of 10 nm particles immobilized on the substrate. The concept and technique of seamless dynamic phase control on the Fourier plane constitute a key asset for modern optical microscopy.