A unique approach toward near-infrared fluorescent probes for bioimaging with remarkably enhanced contrast

A universal sensing platform with significantly enhanced image contrast and apparent brightness in biological imaging applications.


Materials and instruments.
All reagents were purchased from commercial suppliers and used without further purification. Solvents were purified by standard methods. All solutions were prepared using ultrapure water, which was prepared through a Millipore Milli-Q water purification system (Billerica, MA, USA). TLC analysis was performed on silica gel plates, and column chromatography was conducted over silica gel (mesh 200-300), both of which were obtained from Qingdao Ocean Chemicals. 1 H and 13 C NMR spectra were recorded on a Bruker DRX-400 spectrometer operating at 400 and 100 MHz with chemical shifts reported as ppm (in DMSO-d6, TMS as internal standard).
Mass spectrometric data LC-MS analyses were performed using an Agilent 1100 HPLC/MSD spectrometer. The pH measurements were carried out on a Mettler-Toledo Delta 320 pH meter. UVvis absorption spectra were recorded with a Shimadzu UV-2450 spectrophotometer. ºC for 2 h. The absorbance value at 490 nm was determined by a microplate reader.

S6
Preparation and Staining of Rat Liver Tissue Slices. Tissue slices were prepared from rat liver frozen slices. A side of the tissue was cut flat using a vibrating-blade microtome. The slices were cultured with 5 μM HN7, 5 μM Cy5, 10 μM HN7-N2 and then 100 μM NO or 10 μM HN7-S and then 20 μM Hg 2+ in an incubator at 37 °C for 60 min, respectively, followed by washing three times with PBS before imaging. The images were collected in red emission 650-720 nm upon excitation at 635 nm. Scale bar = 120 μm. For comparative experiments, the blank group was anesthetized without any treatment. The second group was given an intraperitoneal injection of Cy5 (5 nanomoles). The third group was given an intraperitoneal injection of HN7 (5 nanomoles).
For analyte detection in vivo, the control group was only given an intraperitoneal injection of probe (HN7-N2 or HN7-S). The second group was given an intraperitoneal injection with probe, followed by intraperitoneal injection with analyte (NO or Hg 2+ ) after 30 min.
Before imaging, the mice were anesthetized by inhalation of 5% isoflurane in 100% oxygen. The animals were placed into the imaging chamber and kept under anesthetic using an isoflurane gas anesthesia system. The nude mice were imaged using a Caliper VIS Lumina XR small animal optical in vivo imaging system. Unless otherwise stated, imaging mode for all experiments was set as excitation scan, and Input/Em was chosen as 605 nm for excitation with Cy5.5 filter for emission channel. All fluorescence images were acquired with autoexposure (FOV, 10 cm; f/stop 2; bin, high resolution), and fluorescence intensity was scaled as units of photons per second per square centimeter per steradian (ps -1 cm -2 sr -1 ).