Increased Depth of Cellular Imaging in the Intact Lung Using Far‐Red and Near‐Infrared Fluorescent Probes

Abstract

Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834) to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm) was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher (27.8 ± 0.7 μm) than for GFP (20 ± 0.5 μm; p = 0.008; n = 50), corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes (36.4 ± 1 μm from the pleural surface) compared with GFP (30.1 ± 0.5 μm; p = 0.01; n = 50). Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs.

Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles

“…The far-red fluorochrome present in DiD (compound 1a) (ε = 225,000 M −1 cm −1 at 650 nm) is an indodicarbocyanine suitable for optical imaging with 633 nm HeNe or 647 nm Kr-Ar lasers and standard Cy5 filters (Ex ~ 650 nm; Em ~ 670 nm) [25]. Reduced autofluorescence in the far red provides improved sensitivity and allows optical imaging at greater depths within tissue [26] than is possible in the visible range. The lipophilic cationic dialkylcarbocyanine fluorophore DiI (compound 1b) (ε = 148,000 M −1 cm −1 at 550 nm) exhibits orange fluorescence and is suitable for optical imaging with 543 HeNe or 561 diode lasers and standard Rhodamine/Cy3 filters (Em 545 /Ex 610 ).…”

Interconnected Cavernous Structure of Bacterial Fruiting Bodies

“…GFP fluorescence uses 480 nm excitation light and emits at a wavelength of 510 nm [14]. As light travels through any media, it undergoes both Raleigh scattering by particles smaller than the wavelength of the light and Mie scattering by particles that larger than the wavelength of light.…”

Early incorporated endothelial cells as origin of metastatic tumor vasculogenesis

“…Lung tumors were evaluated for presence of cells with endothelial phenotype in an experimental metastasis model after tail vein injection of a murine breast adenocarcinoma cell line stably expressing GFP (4T1-GFP) used previously in both experimental and spontaneous metastasis assays [12,13]. This cell line forms solid tumors in the lung with a tumor diameter of up to 500 lm at 7 days post injection.…”

“…A vasculogenic switch, possibly activated by hypoxia at larger tumor sizes, transforms the incorporated endothelial cells into functional blood vessels. cell line stably expressing GFP that we have used previously in both experimental and spontaneous metastasis assays [12,13].…”

“…Experimental metastasis model [12,14,15] Four to 6 week young nu/nu mice (Charles River Lab, Wilmington, MA) were injected into the tail-vein with single-cell suspensions of 4T1-GFP cells at 30 9 10 3 cells per g body weight in 100 lL serum free medium. After 1 and 3 weeks, the mice were sacrificed and lungs with tumors were obtained for immunohistofluorescence with endothelium-specific antibodies and Griffonia simplicifolia lectin labeling of metastatic tumors.…”