RESUMEN
Coherence-controlled holographic microscope (CCHM) combines off-axis holography and an achromatic grating interferometer allowing for the use of light sources of arbitrary degree of temporal and spatial coherence. This results in coherence gating and strong suppression of coherent noise and parasitic interferences enabling CCHM to reach high phase measurement accuracy and imaging quality. The achievable lateral resolution reaches performance of conventional widefield microscopes, which allows resolving up to twice smaller details when compared to typical off-axis setups. Imaging characteristics can be controlled arbitrarily by coherence between two extremes: fully coherent holography and confocal-like incoherent holography. The basic setup parameters are derived and described in detail and experimental validations of imaging characteristics are demonstrated.
Asunto(s)
Holografía/instrumentación , Aumento de la Imagen/métodos , Imagenología Tridimensional/instrumentación , Interferometría/instrumentación , Iluminación/instrumentación , Microscopía/instrumentación , Tomografía de Coherencia Óptica/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
Transmitted-light coherence-controlled holographic microscope (CCHM) based on an off-axis achromatic interferometer allows us to use light sources of arbitrary degree of temporal and spatial coherence. Besides the conventional DHM modes such as quantitative phase contrast imaging and numerical 3D holographic reconstruction it provides high quality (speckle-free) imaging, improved lateral resolution and optical sectioning by coherence gating. Optical setup parameters and their limits for a technical realization are derived and described in detail. To demonstrate the optical sectioning property of the microscope a model sample uncovered and then covered with a diffuser was observed using a low-coherence light source.
Asunto(s)
Holografía/métodos , Microscopía de Interferencia/métodos , Óptica y Fotónica , Algoritmos , Animales , Diseño de Equipo/instrumentación , Humanos , Interpretación de Imagen Asistida por Computador/instrumentación , Interpretación de Imagen Asistida por Computador/métodos , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional/métodos , Interferometría/métodos , Rayos Láser , Microscopía Confocal/métodos , Microscopía de Contraste de Fase/métodos , Nefelometría y TurbidimetríaRESUMEN
We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after approximately 140 min) into the right jugular vein, and the fluorescence signal (at 650-670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 +/- 0.26, mean +/- SE, n = 10), indicating a "priming," or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 +/- 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand beta2-glycoprotein.