Bhattacharya Lab (Lung Biology Lab)

Research

Active Research Projects

Real-Time Confocal Fluorescence Imaging of Alveolar Epithelium and Endothelium

Live views of alveoli and capillaries by confocal microscopy of a mouse lung. Images show the alveolar-capillary region of the lung.

Live views of alveoli and capillaries by confocal microscopy of a mouse lung. Images show the alveolar-capillary region of the lung. [Left:] Green dye shows vessels lying adjacent to an alveolus. [Right:] High magnification shows epithelial cells (red) and an endothelial cell (green).

Imaging Bacterial Dynamics on Alveolar Epithelium

Confocal image of live mouse lung alveoli (alv) shows microaggregates (arrowheads) of GFP-labeled S. aureus strain USA300 (green).

S. aureus USA300 stabilize in lung alveoli by rapidly forming microaggregates in alveolar niches. Confocal image of live mouse lung alveoli (alv) shows microaggregates (arrowheads) of GFP-labeled S. aureus strain USA300 (green). The indicated alveolar septa (i-iii) demarcate an example of calcein-loaded epithelia (red) that converge to form a niche. Scale bar, 20um (Hook, 2018).

Alveolar HCl Causes Transient H2O2-Dependent Mitochondrial Depolarization in Lung Endothelium

Alveolar HCl causes transient H2O2-dependent mitochondrial depolarization in lung endothelium.

Alveolar HCl causes transient H2O2-dependent mitochondrial depolarization in lung endothelium. Confocal images of live mouse lungs at baseline (left column) show calcein green (CG)-labeled alveolar (alv) epithelium (green), calcein red (CR)-labeled capillary (cap) endothelium (red) and potentiometric dye Tetramethylrhodamine ethyl ester (MRE) labelling of lung endothelial mitochondria (pseudo-coloured blue). Alveolar HCL injection caused loss of alveolar CG in 10 mins but not of endothelial CR (middle column).  However, reduced TMRE fluorescence indicates depolarization of endothelial mitochondria. Re-loading TMRE and CG 30 mins after HCL injection (rightmost column) indicate recovery of mitochondrial potential and resealing of the alveolar epithelial plasma membrane. Scale bars, 20um (Hough, 2019).

Molecular Mobility of Cadherin in Endothelial Junctions

Live confocal images of lung endothelial cells show regions of low (LAJ) and high (HAJ) cadherin density.

Live confocal images of lung endothelial cells show regions of low (LAJ) and high (HAJ) cadherin density. By FRAP, the indicated sites (arrows in “bleached” images) were photobleached. Then, the time to fluorescence recovery was quantified by kymograph. The FRAP image shows that recovery was incomplete at 5 min in the HAJ. Thus, junctional proteins have different mobility rates at different regions in endothelial junctions. (Quadri, 2012).

Live Imaging of Alveolar Macrophage

Live confocal image of mouse lung shows sessile alveolar macrophages (arrows) lying adjacent to alveolar septa (red).

Live confocal image of mouse lung shows sessile alveolar macrophages (arrows) lying adjacent to alveolar septa (red). The macrophages communicate with alveolar epithelium through connexin-43 containing gap junctions. The junctions communicate counter-inflammatory calcium signals from macrophages to the epithelium (Westphalen, 2014).

Bone Marrow Stem Cell-Derived Mitochondrial Transfer to Alveolar Epithelial Cells

Image of mitochondrial regulation of acute lung injury.

Mitochondrial regulation of acute lung injury. One of our goals is to understand mitochondrial mechanisms underlying ALI. We reported that mitochondrial transfer from bone marrow-derived mesenchymal stem cells (BMSCs) to injured lung cells (arrows) rescues ALI. Ongoing studies aim at molecular mechanisms underlying the mitochondrial protective effect (Islam, 2012).

Micromechanics of Fluid-Filled Alveoli

3-D alveolar geometry during lung inflation. The effect of lung volume on alveolar geometry impacts several aspects of lung function. Moreover, during lung expansion, the pattern of alveolar perimeter distension is an important determinant of lung functions as, for example, surfactant secretion. Using optical sectioning microscopy, this project aims to correlate inflation-induced changes in alveolar geometry with lung function.

Image of 3-D alveolar geometry during lung inflation.

Alveolar micromechanics. In a single-alveolus model of pulmonary edema, unexpected micromechanical effects ensue when air-filled and liquid-filled (green) alveoli are juxtaposed. The liquid-filled alveolus shrinks imposing mechanical stress on its air-filled neighbor. The mechanical stress reduces compliance of the air-filled alveolus, making it a target for over-expansion injury. (Perlman, 2011).

High Tidal Volume Ventilation Induces Platelet Protein Deposition on Lung Endothelium

Activated platelets deposit proteins on the endothelial surface. Confocal images show freshly isolated single endothelial cells from lungs exposed to low- (LV) or High- (HV) volume ventilation.

Activated platelets deposit proteins on the endothelial surface. Confocal images show freshly isolated single endothelial cells from lungs exposed to low- (LV) or High- (HV) volume ventilation. P-selectin and vWf were deposited on the endothelial surface after HV, not LV. Platelet depletion (PDB) in blood (WB) blocked the effect. Deposition of these proteins provides a platform for coagulation and further injury (Yiming, 2008).

Intra-Nasal GTPase, V12Rac1 Prevents Acute Lung Injury by Enhancing Alveolar F-Actin

A. Images show F-actin levels (rhodamine-phalloidin) of alveolar epithelium 15 mins after alveolar microinfusions of fluorophore-tagged (GFP, green) GTPase Rac1 mutants. Constitutively active V12Rac1 caused rapid and sustained F-actin increase over 4 hours.  In contrast, inactive N17Rac1 had no effect.

B. 6 mice were intra-nasally instilled with PBS (n=3) or non-lethal LPS (n=3) at a dose of 1mg/ml.   In a second instillation 4 hours later, one each of the PBS and LPS recipients was given PBS. Of the remaining PBS (2) and LPS (2) recipients, TAT-conjugated V12Rac1 or N17Rac1 was given to each, as indicated. Images were obtained 24 hours after the first instillation.  LPS increased epithelial TNFR1 (red) expression and neutrophil (Ly6G+) entry, signifying inflammation (leftmost, lower).   V12RAC1 (middle, lower) but not N17RAC1 (right, lower) blocked both inflammatory effects. A and B, Scale bars, 50 μm. (Gusarova, 2021).

Aging-Associated Fibrosis Limits Alveolar Expansion and Surfactant Secretion in Human Lung

Two-photon images show collagen fluorescence by second harmonic generation in the subpleural interstitium of an 18 and an 86 year-old lung. Adjacent panels show collagen fluorescence in the depth plane (y-z) along the indicated lines (dashed lines).

A. Two-photon images (i and ii) show collagen fluorescence by second harmonic generation in the subpleural interstitium of an 18 (left) and an 86 (right) year-old lung. Adjacent panels show collagen fluorescence in the depth plane (y-z) along the indicated lines (dashed lines).

Confocal images show an alveolus (alv) stained with the intracellular dye, calcein-AM. Alveoli were imaged at alveolar pressures 5 (green) and 20 cmH20 (red).

B. Confocal images show an alveolus (alv) stained with the intracellular dye, calcein-AM. Alveoli were imaged at alveolar pressures 5 (green) and 20 cmH20 (red). The alveolar diameter at 5 cmH2O is marked by the line ‘a’. The septal displacement at 20 cmH2O is marked by the line ‘b’ (see magnified image of the region selected by the rectangle).

The images show an alveolus stained with calcein-AM (green) and the extracellular lipid dye FM1-43 (red).

C. The images show an alveolus stained with calcein-AM (green) and the extracellular lipid dye FM1-43 (red). A selected region (rectangle in left image) was magnified in the middle and right images. Alveolar stretch caused surfactant secretion as indicated by time-dependent increase of red fluorescence (arrow).  (Lee, Islam 2021)