mitochondrial physiology | calcium imaging | membrane potential | reactive oxygen species | organelle and cell motility | shape changes in time

Feature highlight: Tools for mitochondrial morphology and dynamics

• Mitochondrial segmentation - menu-accessible pipelines have been optimized for segmentation of images of labeled mitochondria, including fluorescence over inhomogeneous background typical to wide-field  microscopy.
• Follow how morphological parameters, numbers, length, width or roundness change in time. Do it with simple built-in pipelines.
• Quantify mitochondrial biogenesis with mitochondrion:cell volume fraction measurements using confocal microscopy.
• Follow mitochondrial swelling using the thinness ratio technique.
• Quantify mitochondrial motility with the optical flow technique.

See more information in our updated protocols page:  www.imageanalyst.net/protocols.html

Image Analyst Software | 43 Nova Lane, Novato, CA, 94945, USA  | (877)-874-0723 | sales@imageanalyst.net | www.imageanalyst.net

mitochondrial physiology | calcium imaging | membrane potential | reactive oxygen species | organelle and cell motility | shape changes in time

Feature highlight: High pass spatial filtering distinguishes mitochondrially originated fluorescence

• High pass filtering transmits mitochondrial fluorescence (fluorescence originating from thin structures) and blocks cytosolic (bulk) fluorescence
• Optimization module allows automated search for the appropriate filter function
• Combination of high pass filtering with pharmacological calibration allows calculation of mitochondrial and cytosolic NADH/NAD⁺ ratios

Other related functions:

• low light level background subtraction
• image registration
• spectral unmixing
• automatic ROI drawing
• export of intensity traces to text or Excel files

Publication highlight

Just published in Cell Metabolism: Image Analyst MKII was used to extract mitochondrial NAD(P)H fluorescence from time lapses recorded by a Zeiss LSM 7MP two-photon microscope. High pass filtering allowed measurement of the redox states of mitochondrial and cytosolic pyridine nucleotide pools in human fibroblasts. (Wiley et al., Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype. Cell Metab. 2016 Feb 9;23(2):303-14 PMID: 26686024). 

Image Analyst Software | 43 Nova Lane, Novato, CA, 94945, USA  | (877)-874-0723 | sales@imageanalyst.net | www.imageanalyst.net

calcium imaging | membrane potential | reactive oxygen species | organelle and cell motility | shape changes in time

Feature highlight: Analyze calcium imaging data

Analyze image recordings with a single click: built-in pipelines perform:

• background subtraction
• image registration
• spectral unmixing
• automatic ROI drawing
• export of intensity or ratio traces to text or Excel files

Deal with special situations:

• Use mean intensity projection of sub-stacks with Z-drift stabilization and X,Y-image registration to analyze two-photon or confocal microscopic Ca²⁺ imaging data.
• Follow intracellular Ca²⁺ in motile cells with tracking.
• Record large numbers of fluorescence traces by automatic image segmentation and automated, pipeline-based analysis of multi-stage position imaging data.
• Use high-pass filtering to measure fluorescence intensities originating from small or thin objects, like mitochondria.
  

Publication highlight

Just published in Nature: Signal integration by Ca²⁺ regulates intestinal stem-cell activity. (Deng, Gerencser and Jasper, Nature 2015 Epub Dec 2, PMID: 26633624).  Image Analyst MKII was used for determination of GCaMP to mCherry fluorescence ratios in mean intensity projected XYZT-stacks recorded by a Zeiss LSM 7MP two-photon microscope. Image stabilization (registration) and mean intensity Z-projection in Image Analyst MKII enabled efficient use of ROIs to read out single cell fluorescence traces from tissue 3D recordings.

Image Analyst Software | 43 Nova Lane, Novato, CA, 94945, USA  | (877)-874-0723 | sales@imageanalyst.net | www.imageanalyst.net

calcium imaging | membrane potential | reactive oxygen species | organelle and cell motility | shape changes in time

Assay highlight: Image analysis for reactive oxygen species measurements

• Background subtraction algorithms supporting low light level imaging
• Rate measurements - calculate and save rates of fluorescence increase over regions of interests - this is also a useful data reduction step when working with microplate image data
• Spectral unmixing
• Gating for debris and dead cells
• Image registration for gating readout to post-hoc immunocytochemistry

See our protocols for performing assays and image analysis for DCFDA, DHE and MitoSOX: help.imageanalyst.net/protocols.html

Image Analyst Software | 43 Nova Lane, Novato, CA, 94945, USA  | (877)-874-0723 | sales@imageanalyst.net | www.imageanalyst.net

calcium imaging | membrane potential | reactive oxygen species | organelle and cell motility | shape changes in time

Assay highlight: Calculate mitochondrial membrane potential in millivolts in intact cells

Image Analyst MKII provides the complete and extended version of the in situ plasma and mitochondrial membrane potential assay technique developed  by Akos Gerencser and colleagues (PMID: 22495585). The assay is supported by protocols for data acquisition, pipelines for processing image data from a variety of microscopy formats and the intuitive Membrane Potential Calibration Wizard dialog designed for easy and routine use by biologists with no biophysical or mathematical expertise required.

Publication highlight

An application of the absolute mitochondrial membrane potential assay comparing healthy and diseased human samples has been published in the October issue of Endocrinology. The cover page of the journal shows a montage of artificially colored confocal micrographs and processed images used for the calculation of mitochondria:cell volume fractions using Image Analyst MKII. Gerencser AA. Bioenergetic Analysis of Single Pancreatic β-Cells Indicates an Impaired Metabolic Signature in Type 2 Diabetic Subjects. Endocrinology. 2015;156(10):3496–503. PMID: 26204464

Image Analyst Software | 43 Nova Lane, Novato, CA, 94945, USA  | (877)-874-0723 | sales@imageanalyst.net | www.imageanalyst.net