Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation

2008 ◽  
Vol 104 (6) ◽  
pp. 1809-1817 ◽  
Author(s):  
Ivo P. Torres Filho ◽  
James Terner ◽  
Roland N. Pittman ◽  
Elizabeth Proffitt ◽  
Kevin R. Ward
Keyword(s):  
Oxygen Saturation ◽  
Raman Spectra ◽  
Raman Microspectroscopy ◽  
Glass Capillary ◽  
Isolated Cells ◽  
Noninvasive Measurements ◽  
Hb Concentration ◽  
532 Nm

The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So2) in vivo. We implemented a system for in vivo noninvasive measurements of So2. A laser light was focused onto areas of 15–30 μm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So2 was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm−1 range (oxidation state marker band ν4), as well as from the ν19 and ν10 bands (1,500- to 1,650-cm−1 range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary pathlength and Hb concentration did not affect So2 estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So2 estimations and So2 measured by CO-oximetry was highly significant for ν4, ν10, and ν19 bands. The method allowed So2 determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So2 determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.

Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy

2005 ◽  
Vol 289 (1) ◽  
pp. H488-H495 ◽  
Author(s):  
Ivo P. Torres Filho ◽  
James Terner ◽  
Roland N. Pittman ◽  
Leonardo G. Somera ◽  
Kevin R. Ward
Keyword(s):  
Oxygen Saturation ◽  
Raman Spectra ◽  
Raman Band ◽  
Ultraviolet Region ◽  
Excitation Wavelength ◽  
Isolated Cells ◽  
Light Power ◽  
Hemoglobin Oxygen Saturation ◽  
Rat Mesentery

A system is described for in vivo noninvasive measurements of hemoglobin oxygen saturation (HbO2Sat) at the microscopic level. The spectroscopic basis for the application is resonant Raman enhancement of Hb in the violet/ultraviolet region, allowing simultaneous identification of oxy- and deoxyhemoglobin with the same excitation wavelength. The heme vibrational bands are well known, but the technique has never been used to determine microvascular HbO2Sat in vivo. A diode laser light (power: 0.3 mW) was focused onto sample areas 15–30 μm in diameter. Raman spectra were obtained in backscattering geometry by using a microscope coupled to a spectrometer and a cooled detector. Calibration was performed in vitro by using glass capillaries containing blood at several Hb concentrations, equilibrated at various oxygen tensions. HbO2Sat was estimated using the Raman band intensities at 1,360 and 1,375 cm−1. Glass capillary path length and Hb concentration had no effect on HbO2Sat estimated from Raman spectra. In vivo observations were made in blood flowing in microvessels of the rat mesentery. The Hb Raman peaks observed in oxygenated and deoxygenated blood were consistent with earlier Raman studies that used Hb solutions and isolated cells. The method allowed HbO2Sat determinations in the whole range of arterioles, venules, and capillaries. Tissue transillumination allowed diameter and erythrocyte velocity measurements in the same vessels. Raman microspectroscopy offers distinct advantages over other currently used techniques by providing noninvasive and reliable in vivo determinations of HbO2Sat in thin tissues as well as in solid organs and tissues, which are unsuitable for techniques requiring transillumination.

Studies of Metabolism Mediated Mutagenicity In Vitro

1990 ◽  
Vol 18 (1_part_1) ◽  
pp. 243-250
Author(s):  
Dag Jenssen ◽  
Lennart Romert
Keyword(s):  
Cell Lines ◽  
Biological Effects ◽  
Animal Experiments ◽  
Culture Technique ◽  
Organ Perfusion ◽  
Isolated Cells ◽  
Toxicological Effect ◽  
In Vitro Methods

To understand the cause of the biological effects of xenobiotic metabolism in mammals, investigators have traditionally performed animal experiments by comparing the results of biochemical methods, such as measurement of enzyme activity analysis of the metabolites produced, with the observed toxicological effect. This article deals with in vitro methods for genotoxicity combined with drug metabolising preparations at the organelle, cell or organ levels, as exemplified by microsome preparations, isolated cells/cell lines and organ perfusion systems, respectively. The advantage of some of these methods for studying metabolism-mediated mutagenicity is that the measured endpoint reflects not only the bioactivating phase I reactions, but also the detoxifying phase II reactions, and the transfer of the non-conjugated reactive metabolites to other cells and their ability to cause mutations in these cells. In vivo, all these events are important factors in the initiation of cancer. A mechanistic advantage of the methods for metabolism-mediated mutagenicity in vitro is that the relevance of the different steps in metabolism for the mutational events can seldom be investigated in an in vivo assay. Furthermore, human studies can easily be performed using the co-culture technique with isolated human cells or cell lines.

scholarly journals Optical Measurement of Blood Oxygen Saturation of Dental Pulp

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Satoko Kakino ◽  
Shinya Kushibiki ◽  
Azusa Yamada ◽  
Zenzo Miwa ◽  
Yuzo Takagi ◽  
...  
Keyword(s):  
Light Intensity ◽  
Oxygen Saturation ◽  
Dental Pulp ◽  
Optical Measurement ◽  
Blood Oxygen ◽  
Arterial Pulse ◽  
Blood Oxygen Saturation ◽  
Visible Wavelengths

The applicability of arterial pulse oximetry to dental pulp was demonstrated using in vitro and in vivo measurements. First, porcine blood of known oxygen saturation (SO2) was circulated through extracted human upper incisors, while transmitted-light plethysmography was performed using three different visible wavelengths. From the light intensity waveforms measured in vitro, a parameter that is statistically correlated to SO2 was calculated using the pulsatile/nonpulsatile component ratios of two wavelengths for different SO2. Then, values were measured in vivo for living incisors, and the corresponding SO2 values were calculated using the results of in vitro measurements. The estimated SO2 values of the upper central incisors measured in vivo were from 71.0 to 92.7%. This study showed the potential to measure the oxygen saturation changes to identify the sign of pulpal inflammation.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Claudia Noack ◽  
Maria P Zafiriou ◽  
Anke Renger ◽  
Hans J Schaeffer ◽  
Martin W Bergmann ◽  
...  
Keyword(s):  
Cell Fate ◽  
Transcriptional Activation ◽  
Progenitor Cell ◽  
Cellular Localization ◽  
Target Genes ◽  
Critical Role ◽  
Isolated Cells ◽  
Cardiac Progenitor Cell

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs. In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation. In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes. CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype. This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.

In Vivo and In Vitro Studies of a Chronic Oxygen Saturation Sensor

1991 ◽  
Vol 14 (10) ◽  
pp. 1514-1527 ◽  
Author(s):  
GEORGE PHILIP SEIFERT ◽  
ALAN A. MOORE ◽  
KEN L. GRAVES ◽  
STUART P. LAHTINEN
Keyword(s):  
Oxygen Saturation ◽  
In Vitro Studies

scholarly journals Oncogene expression in autoimmune and normal peripheral blood mononuclear cells.

1986 ◽  
Vol 163 (5) ◽  
pp. 1292-1307 ◽  
Author(s):  
D M Klinman ◽  
J F Mushinski ◽  
M Honda ◽  
Y Ishigatsubo ◽  
J D Mountz ◽  
...  
Keyword(s):  
B Cells ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Isolated Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Additional Cell ◽  
Normal Controls

PBMC from patients with autoimmune diseases and from normal controls were studied for the expression of several cellular oncogenes. Gene expression was assessed by Northern blot analysis of poly(A)+ RNA obtained from leukapheresis samples. Patients with SLE expressed significantly more c-myc protooncogene RNA than did normal controls. Increased expression of the N-ras protooncogene was found in that subset of patients whose autoimmune disease was very active. Cells from individuals with SLE, but not from those with other autoimmune illnesses, showed significantly decreased levels of the c-myb and c-fos protooncogenes. To examine the implications of these findings, B and T cells were purified from apheresis samples donated by normal volunteers. When mitogen was used to activate the B cells in vitro, their pattern of protooncogene expression changed to resemble that found in freshly isolated cells from lupus patients. These results suggest that the differences detected in the expression of protooncogenes by patients with SLE may be due to the abnormal activation of their B cells in vivo. The pattern of protooncogene expression found in patients with other autoimmune illnesses is consistent with the activation of additional cell types in those diseases.

scholarly journals Emergence and patterning dynamics of mouse definitive endoderm

2019 ◽  
Author(s):  
Maayan Pour ◽  
Abhishek Sampath Kumar ◽  
Maria Walther ◽  
Lars Wittler ◽  
Alexander Meissner ◽  
...  
Keyword(s):  
Definitive Endoderm ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Isolated Cells ◽  
Temporal Window ◽  
Spatio Temporal ◽  
2D And 3D ◽  
Wnt Signal

AbstractThe segregation of definitive endoderm (DE) from mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE onset has been challenging as it occurs within a narrow spatio-temporal window in the embryo. Here we employ a dual Bra-GFP, Sox17-RFP reporter cell line to study DE onset dynamics. We find Sox17 starts in a few isolated cells in vivo. Using 2D and 3D in vitro models, we show that DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17+ cells. Self-sorting coincides with up-regulation of E-cadherin but is not necessary for DE differentiation or proliferation. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.

Determination of PO2 and its heterogeneity in single capillaries

1996 ◽  
Vol 271 (1) ◽  
pp. H365-H372 ◽  
Author(s):  
L. Zheng ◽  
A. S. Golub ◽  
R. N. Pittman
Keyword(s):  
Time Course ◽  
Oxygen Sensor ◽  
Capillary Tube ◽  
Retractor Muscle ◽  
Glass Capillary ◽  
Phosphorescence Lifetime ◽  
Emission Volume ◽  
Po2 Gradients

We have applied the phosphorescence lifetime technique (Vanderkooi, J. M., G. Maniara, T. J. Green, and D. F. Wilson. J. Biol. Chem. 262: 5476-5482, 1987) to determine oxygen tension in single capillaries of the hamster retractor muscle. Palladium meso-tetra(4-carboxyphenyl)porphine (10 mg/ml, pH 7.40, bound to bovine serum albumin) was used as the phosphorescent oxygen sensor. Our measurement system consisted of a microscope configured for epi-illumination, a strobe flash lamp, a 430-nm bandpass excitation filter, and a 630-nm cut-on emission filter. A rectangular diaphragm was used to limit the illumination field to 10 microns x 10 microns, and an end-window photomultiplier tube was used to detect the phosphorescence signal, which was then input to an analog-to-digital board in a personal computer. In vitro calibrations were carried out at 37 degrees C on samples flowing through a glass capillary tube (diameter, 300 microns) at four different O2 concentrations (0, 2.5, 5, and 7.5%). In vivo tests were carried out on arterioles, capillaries, and venules of the retractor muscle of anesthetized hamsters. The phosphorescent compound was administered by injection into a jugular vein (20 mg/kg). Phosphorescence decay curves were analyzed by a new model of heterogeneous oxygen distribution in the excitation/emission volume. Mean Po2 values and the local Po2 gradients within the excitation/ emission volume were calculated from phosphorescence life-times obtained from individual decay curves. The time course of Po2 obtained during 0.5-s measurement periods (5 decay curves at 0.1-s intervals) at a given site along a capillary indicated the presence of a gradient in Po2 within the plasma space between and near red blood cells. Similar Po2 gradients were also detected in arterioles and venules. Mean Po2 values for arterioles, capillaries, and venules over the 0.5-s observation period were 27 +/- 5, 14 +/- 2, and 11 +/- 3 (SD) mmHg, respectively. The magnitude of the Po2 gradient in the arterioles, capillaries, and venules was 6 +/- 1, 4 +/- 1, and 2 +/- 1 mmHg/micron, respectively.

PHARMACOLOGICAL STUDIES OF PLATELET ANTIAGGREGANTS USING AN IN VITRO MODEL OF PRIMARY HEMOSTASIS.

1987 ◽  
Author(s):  
S Bellucci ◽  
E Cambau ◽  
B Candalot ◽  
J P Caen
Keyword(s):  
Ex Vivo ◽  
Repeated Measurements ◽  
New Drugs ◽  
Type Iii Collagen ◽  
Type I ◽  
Glass Capillary ◽  
New Device ◽  
Primary Hemostasis

We used a new device simulating in vitro primary haemostasis : more precisely the reactivity of blood to collagen and ADP. Thus an artificial vessel was created consisting of two main parts : a glass capillary (ID 140 um, length 16 mm, siliconized) simulating the haemodynamic resistance of an arteriole and an aperture (ID 150 um) reflecting the injured part of a cut arteriole. This aperture was performed in a cellulose acetate filter covered with collagen type I (3 mg/ml) to provide a defined surface for the adhesion of platelets and soaked with ADP in a concentration similar to that of injured endothelial cells (2 x 10-2 M). The mean - sd control values were 110 ± 24 s, 156 -± 40 ul (n = 25) and correlated well with in vivo bleeding time values (p< 0.01). We studied the effect on this test of classical antiaggregant drugs which act on primary hemostasis by different mechanisms of action. Acetylsalycilic acid (Egic laboratories) prolonged this test for concentrations above 10−5 M, ticlopidine (Millot-Solac laboratories) above 3 × 10−4 M, prostacyclin (Wellcome laboratories) above 5 Õ 10−9 M, the synthetic octapeptide LYS-PRO-GLY-GLU-PRO-GLY-PR0-LYS derived from type III collagen (gift from Y. Legrand) above 5 × 10−4 M. We evidenced a synergistic action between collagen octapeptide and ticlopidine. Thus this device permits the screening of new drugs for their effects on primary hemostasis and the study of ex vivo repeated measurements for the monitoring of antiaggregant therapy.

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