FLUORESCENT IMAGES OF MITOCHONDRIAL REDOX STATES IN IN SITU MOUSE HYPOXIC ISCHEMIC INTESTINES

We have imaged mitochondrial oxidation–reduction states by taking a ratio of mitochondrial fluorophores: NADH (reduced nicotinamide adenine dinucleotide) to Fp (oxidized flavoprotein). Although NADH has been investigated for tissue metabolic state in cancer and in oxygen deprived tissues, it alone is not an adequate measure of mitochondrial metabolic state since the NADH signal is altered by dependence on the number of mitochondria and by blood absorption. The redox ratio, NADH/(Fp + NADH), gives a more accurate measure of steady-state tissue metabolism since it is less dependent on mitochondrial number and it compensates effectively for hemodynamic changes. This ratio provides important diagnostic information in living tissues. In this study, the emitted fluorescence of mouse colon in situ is passed through an emission filter wheel and imaged on a CCD camera. Redox ratio images of the healthy and hypoxic mouse intestines clearly showed significant differences. Furthermore, the corrected redox ratio indicated an increase from an average value of 0.51 ± 0.10 in the healthy state to 0.92 ± 0.03 in dead tissue due to severe ischemia (N = 5). We show that the CCD imaging system is capable of displaying the metabolic differences in normal and ischemic tissues as well as quantifying the redox ratio in vivo as a marker of these changes.

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Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166117 ◽

1996 ◽

Vol 54 ◽

pp. 730-731

Author(s):

E. D. Salmon ◽

J. C. Waters ◽

C. Waterman-Storer

Keyword(s):

Imaging System ◽

Ccd Camera ◽

Transmitted Light ◽

Microtubule Assembly ◽

Live Cells ◽

Optical Efficiency ◽

Multiple Band ◽

Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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Measurement of the Optical Properties of Muscle Tissue In Vivo

10.1115/imece2000-2214 ◽

2000 ◽

Author(s):

P. L. Kopsombut ◽

D. Willis ◽

A. E. Schen ◽

L. X. Xu ◽

X. Xu

Keyword(s):

Optical Properties ◽

Transport Theory ◽

Rapid Development ◽

Ccd Camera ◽

High Sensitivity ◽

Biological Tissues ◽

In Vivo Measurement ◽

Living Tissues

Abstract Along with rapid development of diagnostic and therapeutic applications of lasers in medicine, optical properties of various biological tissues have been extensively studied [1]. Most of the studies were performed in vitro owing to the complexity involved in in vivo measurement. To date, it is well understood that living tissue is an absorbing and scattering heterogeneous medium because of its complex structures including blood network. The transport theory cannot be readily used due to the heterogeneity and the absence of the optical properties of living tissues [2]. In this research, we have developed a procedure for measuring the total attenuation coefficient (μ1) of the exteriorized rat 2-D spinotrapezius muscle in the wavelength ranged from 480–560 nm using the collimated light from a Nitrogen-pumped dye laser and a high-sensitivity CCD camera.

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In Situ Image Acquisition and Measurement of Microdroplets Based on Delay Triggering

Micromachines ◽

10.3390/mi10020148 ◽

2019 ◽

Vol 10 (2) ◽

pp. 148

Author(s):

Xuefeng Chang ◽

Kang Zheng ◽

Dan Xie ◽

Xiayun Shu ◽

Keyu Xu ◽

...

Keyword(s):

Light Source ◽

Delay Time ◽

Imaging System ◽

Image Acquisition ◽

Ccd Camera ◽

Time Interval ◽

Charge Coupled Device ◽

Drop On Demand ◽

Relative Standard

An in situ image acquisition apparatus based on delay triggering for visualizing microdroplets formation is described. The imaging system includes a charge-coupled device camera, a motion control card, a driving circuit, a time delay triggering circuit, and a light source. By adjusting the varying trigger delay time which is synchronized with respect to the signal for jetting, the steady sequential images of the droplet flying in free space can be captured real-time by the system. Several image processing steps are taken to measure the diameters and coordinates of the droplets. Also, the jetting speeds can be calculated according to the delay time interval. For glycerin/water (60:40, mass ratio), under the given conditions of the self-made pneumatically diaphragm-driven drop-on-demand inkjet apparatus, the average of diameter and volume are measured as 266.8 μm and 9944 pL, respectively, and the maximum average velocity of the microdroplets is 0.689 m/s. Finally, the imaging system is applied to measure the volume of 200 microsolder balls generated from the inkjet apparatus. The average diameter is 87.96 μm, and the relative standard deviation is 0.83%. The results show good reproducibility. Unlike previous stroboscopic techniques, the present in situ imaging system which is absence of instantaneous high intensity light employs two control signals to stimulate the microdroplet generator and the charge-coupled device (CCD) camera. Hence, the system can avoid the desynchronization problem of signals which control the strobe light-emitting diode (LED) light source and the camera in previous equipment. This technology is a reliable and cost-effective approach for capturing and measuring microdroplets.

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Nutritive value of Trifolium pratense L. for ruminants estimated from in situ ruminal degradation of neutral detergent fibre and in vivo digestibility of organic matter and energy

Czech Journal of Animal Science ◽

10.17221/304/2009-cjas ◽

2010 ◽

Vol 55 (No. 9) ◽

pp. 372-381 ◽

Cited By ~ 3

Author(s):

V. Koukolová ◽

P. Homolka ◽

O. Koukol ◽

F. Jančík

Keyword(s):

Organic Matter ◽

Nutritive Value ◽

Trifolium Pratense ◽

Crude Fibre ◽

Neutral Detergent Fibre ◽

Average Value ◽

Gross Energy ◽

Trifolium Pratense L

Seven clover (Trifolium pratense L.) samples were collected at three different stages of the same sward (first growth (I), n = 3; first regrowth (II), n = 3; second regrowth (III), n = 1) during the growing season from 10<sup>th</sup> of May to 17<sup>th</sup> of August. Samples were analyzed for chemical composition, gross energy (GE) content, in vivo organic matter digestibility (OMD) and gross energy digestibility (GED) in sheep, and in situ rumen degradability of neutral detergent fibre (NDF). The contents of ash, crude protein (CP), crude fibre (CF), NDF, acid detergent fibre (ADF), acid detergent lignin (ADL) and GE were significantly (P < 0.05) affected by the time of cutting. Average values of 119.2, 197.7, 214.1, 400.7, 296.2, 73.8 g/kg of dry matter (DM) and 18.2 MJ/kg of DM were obtained for ash, CP, CF, NDF, ADF, ADL and GE, respectively. In general, OMD and GED decreased as the cutting time progressed, with average values of 72.4% and 70.2%, respectively. Effective ruminal degradability (ED) of NDF generally decreased (P < 0.05) with the increasing date of cutting at each stage, with the values 66.1% (May 10), 63.6% (May 18), 59.2% (May 25), 64.8% (June 29), 57.4% (July 7), 56.9% (July 13) and 51.6% (August 17). In situ measurements were characterised by an average value of 77.1% for the fraction of NDF potentially degradable in the rumen (b), 0.0703 h–1 for the rate constant of disappearance of fraction b (c), and 77.7% for digestible NDF (DNDF).

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Photoinhibitory printing on leaves, visualised by chlorophyll fluorescence imaging and confocal microscopy, is due to diminished fluorescence from grana

Functional Plant Biology ◽

10.1071/pp99077 ◽

1999 ◽

Vol 26 (7) ◽

pp. 717 ◽

Cited By ~ 18

Author(s):

Barry Osmond ◽

Owen Schwartz ◽

Brian Gunning

Keyword(s):

Chlorophyll Fluorescence ◽

Fluorescence Imaging ◽

Imaging System ◽

Confocal Microscope ◽

Chlorophyll Fluorescence Imaging ◽

Grid Pattern ◽

Chlorophyll Autofluorescence ◽

The Stability ◽

Living Tissues

By analogy with the starch printing technique, it was hypothesised that photoinhibition could be used to print images on leaves that would be invisible to the eye, but easily revealed by chlorophyll fluorescence imaging. We first illustrate the process of chlorophyll fluorescence printing on leaves of the shade plant, Cissus rhombifolia, using photographs of artefacts from starch printing experiments in the laboratory of Molisch. We then use portraits of current leaders in chlorophyll fluorescence research to demonstrate the stability of these images in living tissues. Text printing from microfilm of Ewart’s pioneering studies in photoinhibition shows the resolution of the method with the fixed-focus, portable, imaging system used here. The stability of images, as well as quenching analysis of images and of leaves, suggests that localised photoinactivation, rather than sustained photoprotection, is responsible for the detail displayed by fluorescence printing. Electron micrograph positives of stained thylakoids can be printed to create an illusion of what is imagined to be the source of chlorophyll fluorescence at the membrane level. Individual chloroplasts in adjacent cells under the grid pattern of granal stacks printed on leaves were also examined using a confocal microscope. Compared with chloroplasts in the shaded parts of the grid, those in the photoinactivated parts of the grid show greatly reduced chlorophyll autofluorescence. Moreover, these chloroplasts have lost the localised bright fluorescence from grana. Comparisons of fluorescence yields show that relative chlorophyll autofluorescence from grana observed in the confocal microscope parallels that determined in leaves. Our experiments provide direct visual evidence that fluorescence from grana is lost following photoinactivation of photosystem II in vivo.

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In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163903 ◽

1996 ◽

Vol 54 ◽

pp. 288-289

Author(s):

Greg V. Martin ◽

Ann L. Hubbard

Keyword(s):

Three Dimensional ◽

Integral Membrane Proteins ◽

Polarized Secretion ◽

Microscope Images ◽

Computer Aided ◽

Intracellular Organelles ◽

Cytoskeletal Elements ◽

Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

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