[Ultraviolet fluorescence microscopy].
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Fluorescence↗
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[Two-photon laser scanning fluorescence microscopy for functional cellular imaging: Advantages and challenges or One photon is good... but two is better!].
One of the main challenges of modern biochemistry and cell biology is to be able to observe molecular dynamics in their functional context, i.e. in live cells in situ. Thus, being able to track ongoing molecular events with maximal spatial and temporal resolution (within subcellular compartments), while minimizing interference with tissue biology, is key to future developments for in situ imaging. The recent use of non-linear optics approaches in tissue microscopy, made possible in large part by the availability of femtosecond pulse lasers, has allowed major advances on this front that would not have been possible with conventional linear microscopy techniques. Of these approaches, the one that has generated most advances to date is two-photon laser scanning fluorescence microscopy. While this approach does not really provide improved resolution over linear microscopy in non absorbing media, it allows us to exploit a window of low absorbance in live tissue in the near infrared range. The end result is much improved tissue penetration, minimizing unwanted excitation outside the focal area, which yields an effective improvement in resolution and sensitivity. The optical system is also simplified and, more importantly, phototoxicity is reduced. These advantages are at the source of the success of two-photon microscopy for functional cellular imaging in situ. Yet, we still face further challenges, reaching the limits of resolution that conventional optics can offer. Here we review some recent advances in optics/photonics approaches that hold promises to improve our ability to probe the tissue in finer areas, at faster speed, and deeper into the tissue. These include super-resolution techniques, introduction of non paraxial optics in microscopy and use of amplified femtosecond lasers, yielding enhanced spatial and temporal resolution as well as tissue penetration.
Cells↗
[RADIATION-INDUCED CHANGES IN THE BLOOD SEEN IN FLUORESCENCE MICROSCOPY].
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Acridines↗
Localization of the active principles of the male fern (Dryopteris filix-mas (L.) Schott.) by fluorescence microscopy.
The active principles of the male fern, Dryopteris filix-mas (L.) Schott., consisting of a mixture of phloroglucinol derivatives called "crude filicin", are localized by means of fluorescence microscopy under U.V. 365 nm and monochromatic blue 435-490 nm lights. Crude filicin is autofluorescent, and therefore may be easily localized in the internal glandular hairs of the intercellular spaces of rhizome and leaf bases parenchyma, without pre-treatment of sections. Localization is confirmed both morphologically and chemically, and is possible on a fresh material, as well as on a desiccated one. The test allows also a complete anatomical examination of sections, and may be useful, for its easiness and quickness, in further taxonomic or pharmacognostic studies.
Butyrophenones↗
Use of dark-field and fluorescence microscopy to examine extracellular hyaline accumulation in the mouse.
Small foci of diethylstilbestrol-induced extracellular hyaline material were visualized within the mouse uterus utilizing dark-field and fluorescence microscopy following conventional hematoxylin and eosin staining. Hyaline was observed throughout the connective tissue stroma as well as between smooth muscle cells of the myometrium. The use of these techniques permits the rapid examination of small aggregates of hyaline material which are not visible with conventional hematoxylin and eosin staining and eliminates the need for time-consuming and complicated histochemical techniques. The procedures are simple and may also facilitate the quantitative (morphometric) analysis of this material.
Animals↗
Practical uses of acridine orange fluorescence microscopy of centrifuged blood (QBC Malaria Test) and the QBCII Hematology System in patients attending malaria clinics in Thailand.
We evaluated the detection of malaria parasites using acridine orange fluorescence microscopy of centrifuged blood (AOFM/CB or "QBC Malaria Test") at two government malaria clinics in rural Thailand. In a subgroup of the patients, a QBC Hematology System for the determination of complete blood counts was also utilized. A Giemsa-stained thick smear (GTS) reading of 100 (1,000x) microscopic fields was used as standard. The AOFM/CB sensitivities were 97% overall and 95% for P. falciparum (Pf). Sensitivity was lower for P. vivax (Pv) (76%). Pv sensitivity depended largely on ameboid form density. A threshold for AOFM/CB to consistently detect Pv ameboid forms was estimated to be 10/100 WBC (700/microliters blood). AOFM/CB was capable of detecting Pf gametocytes and schizonts more frequently than GTS. The total Pf rings per microliter blood estimated from GTS was highly correlated with the number of Pf rings per Paralens microscopic field (PMF) suggesting that AOFM/CB could be used quantitatively. From a technical standpoint, the rural tropical settings of Thailand in this study were not an obstacle to the use of QBC Hematology. The system was found to be useful in conjunction with AOFM/CB. However, in patients heavily infected with Pf gametocytes of Pv ameboid forms, their total WBC and lymphocyte counts needed to be appropriately corrected. Overall, AOFM/CB appears to be a promising tool for field diagnosis of malaria if it is affordable to developing countries.
Acridine Orange↗
[CONTRIBUTION TO THE STUDY OF PAROTID ADENOLYMPHOMA. ON CYTOCHEMICAL ASPECTS OF A CASE EXAMINED WITH FLUORESCENT MICROSCOPY].
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Adenolymphoma↗
Time-resolved fluorescence microscopy could correct for probe binding while estimating intracellular pH.
Estimation of intracellular pH by fluorescence ratiometry overcomes many of the limitations such as variations in the pathlength of observation and concentration of the probe, light scattering, and photobleaching. However, binding of probes to membranes and macromolecules is generally not taken into account. By using time-resolved fluorescence microscopy on a variety of cell types, we have shown that the dual-emission fluorescent pH probe carboxy SNARF-1 binds to cellular components in significant levels. The bound population could be resolved in the timescale since its fluorescence lifetime (approximately 3 ns) is significantly larger than that of the free probe. Intracellular pH was estimated from the relative amplitudes corresponding to free probes. This procedure was validated in simple model systems where carboxy SNARF-1 was present in solutions of bovine serum albumin. It was shown that the intracellular pH could be overestimated by as much as 1 pH unit in the absence of correction for probe binding.
3T3 Cells↗
Enhanced immunohistochemical detection of autonomic nerve fibers, cytokines and inducible nitric oxide synthase by light and fluorescent microscopy in rat spleen.
We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-beta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-alpha, iNOS, IFN-gamma and c-fos, animals were injected IV with saline or 100 microg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-microm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-gamma, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-alpha, IFN-gamma, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.
Animals↗
Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells.
We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.
Equipment Design↗
Role of fluorescent microscopy in detecting Mycobacterium leprae in tissue sections.
We compared the sensitivity of the fluorescent method with that of he modified Fite-Faraco method in the detection of Mycobacterium leprae in tissue sections. Fifty-six skin biopsies were obtained from patients having leprosy, particularly the paucibacillary type. Minor alterations were made in the deparaffinization and staining technique, as compared with Kuper and May's method, to obtain optimum fluorescence. Of 56 biopsies studied, 39 showed organisms by the fluorescent method and only 25 showed organisms by the modified Fite-Faraco method. The fluorescent method was found to be more advantageous than the modified Fite-Faraco method, particularly in paucibacillary cases. Fluorescent microscopy has the advantage of speed and ease of screening and reduces observer fatigue. Bacillary positivity rates were higher in the fluorescent method than in the modified Fite-Faraco method in each type of leprosy.
Humans↗
Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: calibration of fura-2 ratios.
Isolated rat myocytes incubated with the acetoxy methyl ester of fura 2 contained partially hydrolyzed esters, necessitating in vivo calibration of the signals obtained by fluorescence microscopy for calculation of pCa. Ionophores did not produce reliable R'max and R'min values in respiring myocytes, and elevated free calcium caused individual cells to hypercontract and burst. These difficulties were overcome by superfusion with a glucose-free buffer containing an inhibitor and an uncoupler of oxidative phosphorylation. R'max and R'min values obtained by ionophore treatment of deenergized myocytes were normalized to an in vitro calibration curve. Resting pCa derived from the individual curves averaged 6.9 for calcium-tolerant rod-shaped myocytes.
Adenosine Triphosphate↗
Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy.
Existing methods for studying actin filament dynamics have allowed analysis only of bulk samples or individual filaments after treatment with the drug phalloidin, which perturbs filament dynamics. Total internal reflection fluorescence microscopy with rhodamine-labeled actin allowed us to observe polymerization in real time, without phalloidin. Direct measurements of filament growth confirmed the rate constants measured by electron microscopy and established that rhodamine actin is a kinetically inactive tracer for imaging. In the presence of activated Arp2/3 complex, growing actin filaments form branches at random sites along their sides, rather than preferentially from their barbed ends.
Actin-Related Protein 2↗
Measurement of the effects of acetic acid and extracellular pH on intracellular pH of nonfermenting, individual Saccharomyces cerevisiae cells by fluorescence microscopy.
The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer's wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pHex values was controlled by perfusion with 2 g of total acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pHi of individual S. cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pHex 6.5, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pHi remained constant. The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at pHex 5.6 and 6.5 were significantly smaller than the decreases in pHi at pHex 3.5 and 4.5.
Acetic Acid↗
Subcellular localization of photoreactive ethidium analogs in Trypanosoma brucei by fluorescence microscopy.
To identify the in vivo targets of the trypanocide, ethidium bromide, the fluorescent staining of T. brucei was examined for a series of ethidium analogs using fluorescence microscopy. Determination of the biological targets for most drugs is limited by the reversible nature of their interactions. To overcome this limitation, photoaffinity (azido) analogs of ethidium, which are capable of covalent attachment with photoactivation, were used to identify the ethidium binding sites within the parasites. Two of these compounds, when covalently attached, demonstrated an enhancement of fluorescent staining and were selective for the kinetoplast at low drug concentrations. These compounds were also those found previously to have the highest trypanocidal activity. Propidium, a phenanthridinium analog identical to ethidium except for a larger, more ionic substitution at R5, showed more nonspecific binding as determined by its general staining of the cytoplasm.
Affinity Labels↗
Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide.
Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.
Acridine Orange↗
Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts.
Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64 degrees, both the dorsal and ventral membranes were illuminated, while images excited above 66 degrees illuminated only focal contacts. Above 74 degrees the fluorescence of focal contacts was dominated by back-ground noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 +/- 13 nm. Focal contact areas estimated by TIRFM (1.3 +/- 0.7 micron2) agreed closely with areas observed by immunofluorescence staining of vinculin (1.5 +/- 0.3 microns2).
Animals↗
Novel nontoxic mitochondrial probe for confocal fluorescence microscopy.
We propose a 2,5-Bis[1-(4-N-methylpyridinium)ethen-2-yl)]-N-methylpyrrole ditriflate (PEPEP) as a novel nontoxic, nonpotentiometric mitochondrial probe for confocal fluorescence microscopy. PEPEP is a representative chromophore of a large family of heterocyclic fluorescent dyes that show fluorescence emission in aqueous media and great DNA affinity. We check its cytotoxicity and intracellular localization in mammalian and yeast cell cultures. We demonstrate that PEPEP is a very efficient dye for fluorescence confocal microscopy and a valuable alternative to the most frequently used mitochondrial stains.
Animals↗