• Visualise degranulation at a single cell level and in real-time in vitro and in vivo. 
• Show polarity in the degranulation process. 
• the pH-sensitive impH system shows that a pH shift occurs upon degranulation of pre-formed mediators by activated mast cells.

• Drawbacks with conventional fluorescence microscopy approach – use of fluorescent tag marking specific SG-associated proteins:

• due to transient interaction between, marker proteins and granules, the development of granules cannot be visualised throughout their lifetime. 
• absence of a single or multiple universal markers to label all types of SGs.
• this approach cannot be used to trace/visualise SGs without known markers. ie. without a known origin/ambiguous origin

• Their approach of utilising Negative-Contrast Imaging (NCI) is independent of fluorescently-tagged marker protein, can be used in tandem with a fluorescent probe(s); can be visualised by two-photon microscopy, confocal microscopy, and evanescent-field micrography. They used confocal microscopy in their NCI approach.

Advantages:

• Indirect imaging of secretory granules using GFP-expressing rat mast cells in vitro – produce images showing clearer outlines of individual granules. This is because the approach circumvents the light scattering and diffraction that occurs with the use of fluorescent probes that tend to produce blurry granule boundaries with high fluorescence intensity. 
• using polystyrene beads of 0.5-1 µm diameter, they showed that NCI technique accurately images the SGs. Negatively-stained structures had the theoretical SG diameter of less than 1 µm, enables the visualisation of granule shape, structural and fusion polarity, cell and granule volume, and total SG number in individual cells. 

• Granules are elongated structures along the z-axis and fusion occurs along this axis. 
• Positive correlation between cell volume and granule number. However, smaller granules are observed in cells with higher number of total granules. Their study also showes that the SG volume and number are tightly regulated during mitosis whereby the cell volume varies during cell cycle. This enables the approach to be used in studying cell cycle in mast cells in the setting of allergy and inflammation research area.

• visualise SGs without known markers – more comprehensive visualisation of SGs present in mast cells. When used in combination with fluorescent probes to known SG markers, enables imaging of fusion of unlabelled SG and marked SG (either by Neuropeptide Y or CD63) that can be used to quantify degranulation, study the effects of drugs or molecules regulation mast cell degranulation. 
• Due to its high contrast, enables the construction of crisp 3D images without the concern of photobleaching samples. Illustrated this using the AutoQuantX3 software to deconvolute all images, use ImageJ to convert them into black and white, before incorporating the serial confocal images to construct the 3D image using the Neurolucida software. 

Limitations:

• Limited by miscroscope resolution. Hence, unable to distinguish the cylindrical structures as individual SGs or a cluster of SGs. 
• Without a fluorescent marker, unable to ascertain if the unstained structures are really SGs. Hence, usage is currently best applied in tandem with the conventional fluorescent-tagged molecules to known SG markers. 
• Due to the fast movement of SGs during relocation – within minutes, image quality declines with time-lapse Z-stack scan that require 1-2 minutes per stack for 3D image generation. Thus, needs to use a spinning-disk confocal microscopy to increase image quality.