Near-Infrared Fluorescence Imaging System

What is the Near-Infrared Fluorescence Imaging System?

A near-infrared fluorescence imaging system excites antibodies labelled with 700 and 800 nm fluorophores and detects the emission with matched filters, giving low background multiplexing and a wide linear range for quantitative western blotting.

Overview

Near-infrared imaging uses two lasers or LEDs at about 685 and 785 nm with emission filters near 720 and 820 nm. Membranes scatter and autofluoresce far less in this region than in the visible range, so background is low and two or more targets can be detected simultaneously on the same membrane without stripping and reprobing. Because the signal is proportional to the amount of labelled antibody over a wide range, typically three to four orders of magnitude, quantification is more reliable than with chemiluminescence, which has a narrower linear range. The technique works with both primary antibodies that are directly labelled and with labelled secondary antibodies, and it tolerates repeated imaging because fluorophores do not decay as quickly as chemiluminescent substrates.

How does it work?

Imaging systems convert a signal on a membrane or gel surface into a calibrated digital image. In chemiluminescence imaging, no excitation light is needed: an enzyme such as horseradish peroxidase converts a substrate into an excited product that emits light, and a cooled CCD or CMOS sensor integrates that faint emission over minutes. Fluorescence imaging instead excites fluorophores with LEDs or lasers and collects emitted light through bandpass filters, which allows multiplexing with differently labelled antibodies. Colorimetric systems use white or coloured illumination and measure transmitted or reflected light to quantify stained bands such as Coomassie blue. Laser scanning instruments move a focused beam across the surface and record point by point, which gives high spatial resolution and a wide linear dynamic range. In all cases the software subtracts background, converts pixel intensities to optical density, and compares bands with a standard or loading control.

What are the key features?

• Two near-infrared channels at about 700 and 800 nm
• Low autofluorescence background from membranes and buffers
• Simultaneous detection of two or more targets without stripping
• Linear range of three to four orders of magnitude
• Compatible with direct and secondary antibody labelling
• Repeat imaging without significant signal decay

What are the technical specifications?

A typical near-infrared fluorescence imaging system configuration offers excitation wavelengths of approx. 685 and 785 nm, emission filters of approx. 720 and 820 nm, dynamic range of approx. 3–4 orders of magnitude, detection limit of low femtogram to picogram per band (antibody dependent), scan time of approx. 2–10 min per membrane and imaging area of approx. 100 x 150 mm. Values are indicative of the product class; the exact figures for each configuration are confirmed on the quotation and the calibration documents supplied with the instrument.

Which applications is it used for?

The Near-Infrared Fluorescence Imaging System is used in multiplexed protein detection on one membrane, phospho and total protein ratio measurement, quantitative expression comparison across many samples and loading control normalisation without reprobing. Across the wider Protein Imaging class, laboratories also deploy it for western blot band quantification, protein purity assessment from stained gels and multiplexed fluorescence immunodetection.

Which standards and certifications apply?

Relevant standards and methods include USP <1057>, Ph. Eur. 2.2.31, IEC 61010-1, ISO 17025, 21 CFR Part 11 and ISO 9001. Certification candidates for this model are CE, ISO 9001 manufacturing and IEC 61010-1 safety; each is to be confirmed for the specific configuration before shipment, and certificates are supplied only where they have been issued.

Keep the imaging area clean and free of dust, because particles scatter light and appear as false bands. Avoid saturating the sensor, since saturated pixels cannot be used for quantification; most software flags them, and exposure series should be used to confirm the signal is in the linear range. Chemiluminescent substrates decay within minutes to an hour, so imaging should follow the supplier's timing recommendation and be consistent between samples. Fluorescent blots fade with repeated exposure, so images should be captured once where possible. Calibrate the instrument periodically with a step tablet or fluorescence standard and record the settings used, so that images from different sessions remain comparable. Archive raw files, not only processed figures, because quantitative results may need re-analysis.

How do I select the right Near-Infrared Fluorescence Imaging System?

Select a near-infrared system when multiplexing, wide dynamic range and reproducible quantification are priorities, or when stripping and reprobing would damage the blot. Specify excitation and emission wavelengths, channel count, dynamic range, scan time, membrane compatibility and analysis software.

How does it compare with other Protein Imaging variants?

Choose the detector that matches your detection chemistry. Chemiluminescence needs a cooled sensor with low dark current and long integration times, and absolute sensitivity matters more than resolution. Fluorescence needs matched excitation sources and emission filters, and filter selection should cover the dyes you actually use, including near-infrared pairs for low-background multiplexing. Colorimetric and densitometry work needs uniform illumination and colour calibration to make Coomassie or silver staining quantitative. Laser scanning suits applications where a wide linear dynamic range and high resolution are essential, such as purity determination with small impurity peaks. Check the camera or scanner resolution in millimetres per pixel, dynamic range in orders of magnitude, cooling temperature, imaging area, light source lifetime and whether the software supports background subtraction, saturation warnings, lane profiling and export of numerical data.