RESUMO
Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.
Assuntos
Células-Tronco Hematopoéticas , Humanos , Camundongos , Animais , Células-Tronco Hematopoéticas/metabolismo , Concentração de Íons de HidrogênioRESUMO
In this study, we characterize age-related phenotypes of human hematopoietic stem cells (HSC). We report increased frequencies of HSC, hematopoietic progenitor cells and lineage negative cells in the elderly but a decreased frequency of multi-lymphoid progenitors. Aged human HSC further exhibited a delay in initiating division ex vivo though without changes in their division kinetics. The activity of the small RhoGTPase Cdc42 was elevated in aged human hematopoietic cells and we identified a positive correlation between Cdc42 activity and the frequency of HSC upon aging. The frequency of human HSC polar for polarity proteins was, similar to the mouse, decreased upon aging, while inhibition of Cdc42 activity via the specific pharmacological inhibitor of Cdc42 activity, CASIN, resulted in re-polarization of aged human HSC with respect to Cdc42. Elevated activity of Cdc42 in aged HSC thus contributed to age-related changes in HSC. Xenotransplant, using NBSGW mice as recipients, showed elevated chimerism in recipients of aged compared to young HSC. Aged HSC treated with CASIN ex vivo displayed an engraftment profile similar to recipients of young HSC. Taken together, our work reveals strong evidence for a role of elevated Cdc42 activity in driving aging of human HSC, and similar to mice, this presents a likely possibility for attenuation of aging in human HSC.
Assuntos
Envelhecimento , Células-Tronco Hematopoéticas , Idoso , Animais , Células-Tronco Hematopoéticas/metabolismo , Humanos , CamundongosRESUMO
Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain homeostasis. With aging, the frequency of polar HSCs decreases. Cell polarity in HSCs is controlled by the activity of the small RhoGTPase cell division control protein 42 (Cdc42). Here we demonstrate-using a comprehensive set of paired daughter cell analyses that include single-cell 3D confocal imaging, single-cell transplants, single-cell RNA-seq, and single-cell transposase-accessible chromatin sequencing (ATAC-seq)-that the outcome of HSC divisions is strongly linked to the polarity status before mitosis, which is in turn determined by the level of the activity Cdc42 in stem cells. Aged apolar HSCs undergo preferentially self-renewing symmetric divisions, resulting in daughter stem cells with reduced regenerative capacity and lymphoid potential, while young polar HSCs undergo preferentially asymmetric divisions. Mathematical modeling in combination with experimental data implies a mechanistic role of the asymmetric sorting of Cdc42 in determining the potential of daughter cells via epigenetic mechanisms. Therefore, molecules that control HSC polarity might serve as modulators of the mode of stem cell division regulating the potential of daughter cells.
Assuntos
Divisão Celular/genética , Senescência Celular/genética , Epigênese Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Envelhecimento/metabolismo , Animais , Divisão Celular Assimétrica/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Agregação Celular , Linhagem da Célula/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Cromatina , Camundongos Endogâmicos C57BL , Transcriptoma/genética , Proteína Wnt-5a/farmacologia , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Human plasmacytoid dendritic cells (pDC) are important modulators of adaptive T cell responses during viral infections. Recently, we found that human pDC produce the serine protease granzyme B (GrB), thereby regulating T cell proliferation in a GrB-dependent manner. In this study, we demonstrate that intrinsic GrB production by pDC is significantly inhibited in vitro and in vivo by clinically used vaccines against viral infections such as tick-borne encephalitis. We show that pDC GrB levels inversely correlate with the proliferative response of coincubated T cells and that GrB suppression by a specific Ab or a GrB substrate inhibitor results in enhanced T cell proliferation, suggesting a predominant role of GrB in pDC-dependent T cell licensing. Functionally, we demonstrate that GrB(high) but not GrB(low) pDC transfer GrB to T cells and may degrade the ζ-chain of the TCR in a GrB-dependent fashion, thereby providing a possible explanation for the observed T cell suppression by GrB-expressing pDC. Modulation of pDC-derived GrB activity represents a previously unknown mechanism by which both antiviral and vaccine-induced T cell responses may be regulated in vivo. Our results provide novel insights into pDC biology during vaccinations and may contribute to an improvement of prophylactic and therapeutic vaccines.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Granzimas/metabolismo , Vacinas Virais/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Encefalite Transmitida por Carrapatos/imunologia , Humanos , Interferon-alfa/biossíntese , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fields, are combined with self-interference digital holographic microscopy. This biophotonic holographic workstation enables non-contact, minimally invasive, flexible, high-precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a first model experiment, internalized polystyrene microspheres with 1 µm diameter are three-dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.
Assuntos
Células , Biofísica , Corantes Fluorescentes , Osmose , FagocitoseRESUMO
Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB(+) pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC-T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.
Assuntos
Artrite Juvenil/imunologia , Células Dendríticas/metabolismo , Granzimas/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/imunologia , Artrite Juvenil/patologia , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Criança , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interleucina-3/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
Plasmacytoid dendritic cell (PDC)-derived IFN-alpha plays a central role in antiviral defense and in Th1-driven autoimmune diseases, such as systemic lupus erythematosus (SLE). In the current study, we explored how PGE2 effects the phenotype of PDCs from healthy and SLE subjects. Although PGE2 is considered to mediate mainly proinflammatory effects, we show that PGE2 and PG analogs potently inhibit secretion of IFN-alpha by TLR-activated PDCs. This effect is mainly mediated by PG receptors E-prostanoid 2 and E-prostanoid 4 and involves inhibition of IFN regulatory factor 7 expression. Of note, profound IFN-alpha inhibition by PGE2 is also seen in PDCs from SLE subjects, independent of age, disease activity, and therapy. We show that TLR9-activated PDCs treated with PGE2 exhibit DC2-like characteristics with enhanced expression of CD86 and CD62L, and decreased expression of CD80 and MHC class I. Consequently, PGE2-treated PDCs suppress secretion of Th1 cytokines by T cells while increasing the secretion of Th2 cytokines. Prevention of CpG-induced CD62L downregulation by PGE2 suggests that it may induce the retreat of PDCs from inflamed tissues. Our data on the effects of PGE2 on PDCs may explain occasional reports about the induction of SLE-like symptoms by cyclooxygenase inhibitors as well as improvement of such symptoms by treatment with PG analogs. In conclusion, our data suggest that PGE2 and certain PG analogs, some of which are already in clinical use, should be evaluated as a novel and inexpensive treatment approach for patients with SLE and other IFN-alpha-dependent, Th1-driven autoimmune diseases.
Assuntos
Células Dendríticas/imunologia , Dinoprostona/fisiologia , Interferon-alfa/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Prostaglandina E/metabolismo , Células Th1/metabolismo , Estudos de Casos e Controles , Citocinas/metabolismo , Dinoprostona/metabolismo , Humanos , Interferon-alfa/metabolismo , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4RESUMO
Quantitative 3D imaging of organ-wide cellular and subcellular components is central for revealing and understanding complex interactions between stem cells and their microenvironment. Here, we present a gentle but fast whole-mount immunofluorescence staining protocol for 3D confocal microscopy (iFAST3D) that preserves the 3D structure of the entire tissue and that of subcellular structures with high fidelity. The iFAST3D protocol enables reproducible and high-resolution 3D imaging of stem cells and various niche components for many mouse organs and tissues. For complete details on the use and execution of this protocol, please refer to Saçma et al. (2019).
Assuntos
Imageamento Tridimensional , Células-Tronco , Animais , Imageamento Tridimensional/métodos , Camundongos , Microscopia Confocal/métodos , Coloração e RotulagemRESUMO
One goal of regenerative medicine is to rejuvenate tissues and extend lifespan by restoring the function of endogenous aged stem cells. However, evidence that somatic stem cells can be targeted in vivo to extend lifespan is still lacking. Here, we demonstrate that after a short systemic treatment with a specific inhibitor of the small RhoGTPase Cdc42 (CASIN), transplanting aged hematopoietic stem cells (HSCs) from treated mice is sufficient to extend the healthspan and lifespan of aged immunocompromised mice without additional treatment. In detail, we show that systemic CASIN treatment improves strength and endurance of aged mice by increasing the myogenic regenerative potential of aged skeletal muscle stem cells. Further, we show that CASIN modifies niche localization and H4K16ac polarity of HSCs in vivo. Single-cell profiling reveals changes in HSC transcriptome, which underlie enhanced lymphoid and regenerative capacity in serial transplantation assays. Overall, we provide proof-of-concept evidence that a short systemic treatment to decrease Cdc42 activity improves the regenerative capacity of different endogenous aged stem cells in vivo, and that rejuvenated HSCs exert a broad systemic effect sufficient to extend murine health- and lifespan.
RESUMO
Hematopoietic stem cells (HSCs) mediate regeneration of the hematopoietic system following injury, such as following infection or inflammation. These challenges impair HSC function, but whether this functional impairment extends beyond the duration of inflammatory exposure is unknown. Unexpectedly, we observed an irreversible depletion of functional HSCs following challenge with inflammation or bacterial infection, with no evidence of any recovery up to 1 year afterward. HSCs from challenged mice demonstrated multiple cellular and molecular features of accelerated aging and developed clinically relevant blood and bone marrow phenotypes not normally observed in aged laboratory mice but commonly seen in elderly humans. In vivo HSC self-renewal divisions were absent or extremely rare during both challenge and recovery periods. The progressive, irreversible attrition of HSC function demonstrates that temporally discrete inflammatory events elicit a cumulative inhibitory effect on HSCs. This work positions early/mid-life inflammation as a mediator of lifelong defects in tissue maintenance and regeneration.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Idoso , Envelhecimento , Animais , Medula Óssea , Humanos , Inflamação , CamundongosRESUMO
Cdc42 is a small RhoGTPase regulating multiple functions in eukaryotic cells. The activity of Cdc42 is significantly elevated in several tissues of aged mice, while the Cdc42 gain-of-activity mouse model presents with a premature aging-like phenotype and with decreased lifespan. These data suggest a causal connection between elevated activity of Cdc42, aging, and reduced lifespan. Here, we demonstrate that systemic treatment of aged (75-week-old) female C57BL/6 mice with a Cdc42 activity-specific inhibitor (CASIN) for 4 consecutive days significantly extends average and maximum lifespan. Moreover, aged CASIN-treated animals displayed a youthful level of the aging-associated cytokines IL-1ß, IL-1α, and INFγ in serum and a significantly younger epigenetic clock as based on DNA methylation levels in blood cells. Overall, our data show that systemic administration of CASIN to reduce Cdc42 activity in aged mice extends murine lifespan.
Assuntos
Citocinas/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Envelhecimento , Animais , Proteínas de Drosophila , Feminino , Cadeias alfa de Integrinas , Longevidade , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Digital holographic microscopy (DHM) enables a quantitative multifocus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms enables subsequent automated focus realignment by numerical propagation of the digital holographically reconstructed object wave. In combination with a calibrated optical imaging system, the obtained propagation data quantify axial displacements of the investigated sample. The evaluation of quantitative DHM phase contrast images also enables an effective determination of lateral cell displacements. Thus, 3-D displacement data are provided. Results from investigations on sedimenting red blood cells and HT-1080 fibrosarcoma cells in a collagen tissue model demonstrate that DHM enables marker-free automated quantitative dynamic 3-D cell tracking without mechanical focus adjustment.
Assuntos
Eritrócitos/citologia , Eritrócitos/fisiologia , Holografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Reconhecimento Automatizado de Padrão/métodos , Algoritmos , Inteligência Artificial , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por ComputadorRESUMO
With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing.
Assuntos
Envelhecimento/genética , Capilares/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Homeostase/genética , Nicho de Células-Tronco/genética , Envelhecimento/metabolismo , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Capilares/citologia , Capilares/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Rastreamento de Células/métodos , Doxiciclina/farmacologia , Fluoruracila/farmacologia , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Homeostase/efeitos dos fármacos , Proteína Jagged-2/genética , Proteína Jagged-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agonistas Mieloablativos/farmacologia , Nicho de Células-Tronco/efeitos dos fármacosRESUMO
BACKGROUND: The decline of hematopoietic stem cell (HSC) function upon aging contributes to aging-associated immune remodeling and leukemia pathogenesis. Aged HSCs show changes to their epigenome, such as alterations in DNA methylation and histone methylation and acetylation landscapes. We previously showed a correlation between high Cdc42 activity in aged HSCs and the loss of intranuclear epigenetic polarity, or epipolarity, as indicated by the specific distribution of H4K16ac. RESULTS: Here, we show that not all histone modifications display a polar localization and that a reduction in H4K16ac amount and loss of epipolarity are specific to aged HSCs. Increasing the levels of H4K16ac is not sufficient to restore polarity in aged HSCs and the restoration of HSC function. The changes in H4K16ac upon aging and rejuvenation of HSCs are correlated with a change in chromosome 11 architecture and alterations in nuclear volume and shape. Surprisingly, by taking advantage of knockout mouse models, we demonstrate that increased Cdc42 activity levels correlate with the repression of the nuclear envelope protein LaminA/C, which controls chromosome 11 distribution, H4K16ac polarity, and nuclear volume and shape in aged HSCs. CONCLUSIONS: Collectively, our data show that chromatin architecture changes in aged stem cells are reversible by decreasing the levels of Cdc42 activity, revealing an unanticipated way to pharmacologically target LaminA/C expression and revert alterations of the epigenetic architecture in aged HSCs.
Assuntos
Senescência Celular , Cromatina , Epigênese Genética , Células-Tronco Hematopoéticas/fisiologia , Lamina Tipo A/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Animais , Metilação de DNA , Feminino , Células-Tronco Hematopoéticas/citologia , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Estrogen receptor (ER) beta gene codes for different transcript variants resulting from alternative splicing. In this study, we report identification of the two novel human exon-skipped ERbeta transcript isoforms ERbetaDelta125 and ERbetaDelta1256 in MDA-MD-231 breast cancer cells. Both transcripts could also be detected in a variety of human tissues. We further report the results of an in vitro attempt to characterize their function in regulation of cell growth, motility, apoptosis and gene expression. COS-1 cells stably transfected with the novel ERbeta transcripts exhibited a notably slower growth even in the absence of estradiol when compared to vector-transfected control cells. Like ERbeta1, both novel ERbeta transcript isoforms raised the basal apoptosis rate of COS-1 cells in a ligand-independent manner. Whereas introduction of ERbetaDelta1256 notably increased the sensitivity of COS-1 cells towards lower concentrations of selective estrogen receptor modulator tamoxifen, presence of ERbeta1 and ERbetaDelta125 transcripts further weakened the growth-inhibitory effect of tamoxifen on this cell line. Furthermore, expression of ERbetaDelta1256 variant was demonstrated to reduce transcript levels of estrogen-responsive genes like cyclin A2, IGFBP-4 and fibulin 1c in COS-1 cells in a ligand-independent manner. Though we were not able to detect the predicted 29 and 34kDa proteins by means of western blot analysis, our data strongly suggest the biological functionality of both isoforms on molecular level. With this report increasing the multitude of existing ERbeta mRNA isoforms, we provide further evidence that their synthesis has to be considered as an important level of estrogen signaling.
Assuntos
Processamento Alternativo , Apoptose , Estradiol/farmacologia , Receptor beta de Estrogênio/biossíntese , Regulação da Expressão Gênica , Transdução de Sinais , Processamento Alternativo/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Isoformas de Proteínas/biossíntese , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Self-interference digital holographic microscopy (DHM) has been found particular suitable for simplified quantitative phase imaging of living cells. However, a main drawback of the self-interference DHM principle are scattering patterns that are induced by the coherent nature of the laser light which affect the resolution for detection of optical path length changes. We present a simple and efficient technique for the reduction of coherent disturbances in quantitative phase images. Therefore, amplitude and phase of the sample illumination are modulated by an electrically focus tunable lens. The proposed method is in particular convenient with the self-interference DHM concept. Results from the characterization of the method show that a reduction of coherence induced disturbances up to 70 percent can be achieved. Finally, the performance for enhanced quantitative imaging of living cells is demonstrated.
RESUMO
A successful application of self-interference digital holographic microscopy in combination with a sample-rotation-based tomography module for three-dimensional (3-D) label-free quantitative live cell imaging with subcellular resolution is demonstrated. By means of implementation of a hollow optical fiber as the sample cuvette, the observation of living cells in different 3-D matrices is enabled. The fiber delivers a stable and accurate rotation of a cell or cell cluster, providing quantitative phase data for tomographic reconstruction of the 3-D refractive index distribution with an isotropic spatial resolution. We demonstrate that it is possible to clearly distinguish and quantitatively analyze several cells grouped in a "3-D cluster" as well as subcellular organelles like the nucleoli and local internal refractive index changes.
Assuntos
Técnicas de Cultura de Células/métodos , Holografia/métodos , Tomografia Óptica/métodos , Linhagem Celular Tumoral , Desenho de Equipamento , Humanos , RefratometriaRESUMO
The analysis of dynamic interactions of microorganisms with a host cell is of utmost importance for understanding infection processes. We present a biophotonic holographic workstation that allows optical manipulation of bacteria by holographic optical tweezers and simultaneously monitoring of dynamic processes with quantitative multi-focus phase imaging based on self-interference digital holographic microscopy. Our results show that several bacterial cells, even with non-spherical shape, can be aligned precisely on the surface of living host cells and localized reproducibly in three dimensions. In this way a new label-free multipurpose device for modelling and quantitative analysis of infection scenarios at the single cell level is provided.
Assuntos
Bacillus subtilis/citologia , Holografia/métodos , Microscopia/métodos , Modelos Biológicos , Pinças Ópticas , Análise de Célula Única/métodos , Bacillus subtilis/fisiologia , Linhagem Celular Tumoral , HumanosRESUMO
The intracellular refractive index is an important parameter that describes the optical density of the cytoplasm and the concentration of the intracellular solutes. The refractive index of adherently grown cells is difficult to access. We present a method in which silica microspheres in living cells are used to determine the cytoplasm refractive index with quantitative phase microscopy. The reliability of our approach for refractive index retrieval is shown by data from a comparative study on osmotically stimulated adherent and suspended human pancreatic tumor cells. Results from adherent human fibro sarcoma cells demonstrate the capability of the method for sensing of dynamic refractive index changes and its usage with microfluidics.
Assuntos
Adesão Celular/fisiologia , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Microscopia de Contraste de Fase/métodos , Microesferas , Refratometria/métodos , Linhagem Celular Tumoral , Meios de Contraste , Humanos , Sondas Moleculares/químicaRESUMO
Many interferometry-based quantitative phase contrast imaging techniques require a separately generated coherent reference wave. This results in a low phase stability and the demand for a precise adjustment of the intensity ratio between object and reference wave. To overcome these problems, the performance of a Michelson interferometer approach for digital holographic microscopy was analyzed that avoids a separately generated reference wave by superposition of different image areas. It is shown that this simplified arrangement yields improved phase stability. Furthermore, results from time-lapse investigations on living pancreas tumor cells demonstrate the capability of the method for reliable quantitative phase contrast imaging.